Molecular constructs with a carcinoembryonic antigen (cea) transcriptional regulatory sequence

ABSTRACT

Novel molecular chimaeras produced by recombinant DNA techniques are described. They comprise a target-tissue specific transcriptional regulatory sequence (TRS) linked and controlling the expression of a heterologous enzyme, for example Varicella Zoster Virus Thymidine Kinase (VZV TK) or non-mammaliam Cytosine Deaminase (CD). A molecular chimaera is packaged into a synthetic retroviral particle that is capable of infecting mammalian tissue. This, in turn, may be administered to a host, and the TRS will be selectively transcriptionally activated in the target tissue (for example cancerous cells). Administration of compounds that are selectively metabolised by the enzyme produce cytotoxic or cytostatic metabolites in situ thereby selectively killing or arresting the growth of the target cells.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 07/841,961 filed filed Feb. 26, 1992 which is acontinuation-in-part of U.S. application Ser. No. 07/662,222 filed Feb.22, 1991 which is a continuation-in-part of U.S. application Ser. No.07/574,994 filed Aug. 29, 1990.

FIELD OF THE INVENTION

[0002] The present invention relates to molecular chimaeras in infectivevirions: methods of their construction; pharmaceutical formulationscontaining them; their use in therapy, particularly virus-directedenzyme prodrug therapy, particularly in the treatment of cancers, andmore particularly in the treatment of hepatocellular and colorectalcarcinomas; and the use of agents which can be catalysed by aheterologous enzyme to cytotoxic or cytostatic metabolites, such aspurine arabinosides and substituted pyrimidines and cytosines invirus-directed enzyme prodrug therapy in a host (e.g., mammal or human).

BACKGROUND OF THE INVENTION

[0003] Cancer of all forms is one of the major causes of morbiditythroughout the world. Research in cancer chemotherapy has produced avariety of antitumour agents with differing degrees of efficacy.Standard clinically used agents include adriamycin, actinomycin D,methotrexate, 5-fluorouracil, cisplatin, vincristine and vinblastine.However, these presently available antitumour agents are known to havevarious disadvantages such as toxicity to healthy cells and resistanceof certain tumour types. Other forms of therapy, such as surgery, areknown. However it is appreciated by those skilled in the art that novelapproaches and entities for cancer therapy are still needed.

[0004] Hepatocellular carcinoma (HCC) is one of the major malignantdiseases in the world today; the greatest incidence being in Japan,China, other parts of Asia, and sub-Saharan Africa. Recent evidencesuggests that the incidence of hepatocellular carcinoma in Europe andNorth America is increasing. The disease is estimated to be responsiblefor or involved in up to approximately 1,250,000 deaths a year, makingit one of the world's major malignant diseases.

[0005] The prognosis of HCC is always poor, with the worldwide frequencyrate almost equalling the mortality rate. After diagnosis, the mediansurvival time is less than four months. Long-term survival, defined assurvival longer than one year after diagnosis, is seen onlyoccasionally. Most HCC patients succumb to either the complications ofliver failure with or without massive bleeding, or to the generaleffects of a large tumour burden, with cachexia, malnutrition,infection, and sepsis. Though distant metastases occur (up to 90% ofpatients have metastatic tumour at time of death), regional disease mostoften limits survival. Consequently, therapies directed toward controlof hepatic tumours are appropriate, although it will be appreciated thattreatment of the metastatic disease is also of great importance (Kew M.C. Postgrad. Med. J. 59 (Suppl. 4) 78-87 (1983) and Berk P. (Ed) Semin.Liver Dis. 4, No.2, Thieme-Stratton Inc. N.Y. (1984)).

[0006] Current therapies available to the clinician are basicallyineffective as a curative treatment for this disease (Nerenstone S. R.,Ihde D. C., Friedman M. A. Cancer Treat. Rev. 15, 1-31 (1988)). To date,surgery continues to be the only potentially curative treatment.However, at the time of diagnosis, the overwhelming majority of patientsare not able to undergo radical surgery. In certain studies (Nerenstoneet al supra) less than 3% of patients were considered capable ofundergoing surgery and of the small percentage that do undergo surgery,approximately 50% suffer from postoperative morbidity (Nerenstone et alsupra).

[0007] Colorectal carcinoma (CRC) is the second most frequent cancer andthe second leading cause of cancer-associated deaths in the UnitedStates and Western Europe (Silverberg, E. CA 33, 9-25(1983); Silverberg,E. CA 36, 9-25(1986); Farley, P. C., and McFaden, K. H. Postgrad. Med.84, 175-183) (1988). The overall five-year survival rate for patientshas not meaningfully improved in the last three decades. Prognosis forthe CRC cancer patient is associated with the depth of tumor penetrationinto the bowel wall, the presense of regional lymph node involvementand, most importantly, the presense of distant metastases. The liver isthe most common site for distant metastasis and, in approximately 30% ofpatients, the sole initial site of tumor recurrence after successfulresection of the primary colon cancer (Daly, J. M., and Kemeny, N.Import. Adv. Oncol. 251-286(1986)). Hepatic metastases are the mostcommon cause of death in the CRC cancer patient (Swinton, N. W., et al.,Dis. Colon Rectum 7, 273-277(1964)).

[0008] The treatment of choice for the majority of patients with hepaticCRC metastasis is systemic or regional chemotherapy using 5-fluorouracil(5-FU) alone or in combination with other agents such as leviamasole(for a review see Daly, J. M., and Kemeny, N. (1986) Import. Adv. Oncol.251-286). However, despite extensive effort, there is still nosatisfactory treatment for hepatic CRC metastasis.

[0009] Systemic single- and combination-agent chemotherapy and radiationare relatively ineffective emphasizing the need for new approaches andtherapies for the treatment of these diseases.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1: Schematic representation of albumin transcriptionalregulatory sequences (TRS) in relation to the albumin gene (1A), and aheterologous gene (1B). The truncated albumin TRS in relation to aheterologous gene (1C).

[0011]FIG. 2A: Diagram of varicella zoster thymidine kinase gene.

[0012]FIG. 2B: VZV TK gene—1^(o) sequence (SEQ ID NO:1).

[0013]FIG. 3A: Albumin transcriptional regulatory sequence/VZV TKmolecular chimaera.

[0014]FIG. 3B: Alpha-fetoprotein transcriptional regulatory sequence/VZVTK molecular chimaera.

[0015]FIG. 4A: Proviral form of retrovirus containingalpha-fetoprotein/VZV TK molecular chimaera.

[0016]FIG. 4B: pCR78.

[0017]FIG. 5: Flow chart showing the construction of pCR74.

[0018]FIG. 6: Flow chart showing the construction of pCR78.

[0019]FIG. 7: Sequence flanking ALB E/P to VZV TK in pCR74(SEQ ID NO:2).E=Enhancer, P=Promoter.

[0020]FIG. 8: Sequence flanking AFP E/P to VZV TK in pCR78 (SEQ IDNO:3). E=Enhancer, P=Promoter.

[0021]FIG. 9: Production of ara-ATP with cells infected with controls,pCR74, and pCR78.

[0022]FIG. 10: Diagram of CEA phage clones. The overlapping cloneslambdaCEA1, iambdaCEA7, and lambdaCEA5 represent an approximately 26 kbregion of CEA genomic sequence. The 11,288 bp HindIII-Sau3A fragmentthat was sequenced is represented by the heavy line under lambdaCEA1.The 3774 bp Hind III-Hind III fragment that was sequenced is representedby the heavy line under lambdaCEA7. The bent arrows represent thetranscription start point for CEA mRNA. The straight arrows representthe oligonucleotides CR15 and CR16. H, HindIII; S, SstI; B, BamHI; E,EcoRI; X, XbaI.

[0023]FIG. 11: Restriction map of part of lambda CEA1. The arrow headrepresents the approximate location of the transcription initation pointfor CEA mRNA. Lines below the map represent the CEA inserts ofpBS+subclones. These subclones are convenient sources for numerous CEArestriction fragments.

[0024]FIG. 12A: DNA sequence of the 11,288 bp HindIII to Sau3A fragmentof lambda CEA7(SEQ ID NO:4). Sequence is numbered with the approximatetranscription initation point for CEA mRNA as 0 (this start site isapproximate because there is some slight variability in the start siteamong indiviual CEA transcripts). The translation of the first exon isshown. Intron 1 extends from +172 to beyond +592. Several restrictionsites are shown above the sequence. In subclone 109-3 the sequence atpositions +70 has been altered by site-directed mutagenesis in order tointroduce HindIII and EcoRI restriction sites.

[0025]FIG. 12B: DNA sequence of the 3774 bp Hind III to Hind IIIfragment of lambda CEA7 (SEQ ID NO:5). Sequence is numbered as in FIG.12A.

[0026]FIG. 12C: Mapplot of 15,056 bp Hind III to Sau 3A fragment fromCEA genomic DNA showing consensus sequences. Schematic representation ofsome of the consensus sequences found in the CEA sequence of FIG. 12Aand 12B. The consensus sequences shown here are from the transcriptionaldictionary of Locker and Buzard (DNA Sequence 1, 3-11 (1990)). Thelysozymal silencer is coded B18. The last line represents 90% homologyto the topoisomerase II cleavage consensus.

[0027]FIG. 13: Cloning scheme for CEA constructs extending from −299 bpto +69 bp.

[0028]FIG. 14A: Cloning scheme for CEA constructs extending from −10.7kb to +69 bp.

[0029]FIG. 14B: Coordinates for CEA sequence present in severalCEA/luciferase clones. CEA sequences were cloned into the multiplecloning region of pGL2-Basic (Promega Corp.) by standard techniques. CEAcoordinates determined using base numbering of FIG. 12A and 12B.

[0030]FIG. 14C: Transient luciferase assays. Transient and 14Dtransfections and luciferase assays were performed in quadruplicate bystandard techniques using DOTAP (Boehringer Mannheim, Indianpolis,Ind.), luciferase assay system (Promega, Madison, Wis.), and Dynatechluminometer (Chantilly, Va.). CEA-positive cell lines included LoVo(ATCC #CCL 229) and SW1463 (ATCC #CCL 234). CEA-negative cell linesincluded HuH7 and Hep3B (ATCC #HB 8064). C. Luciferase activityexpressed as the percent of pGL2-Control plasmid activity. D. Luciferaseactivities of LoVo and SW1463 expressed as fold increase over activityin Hep3B.

[0031]FIG. 15A: An illustration of the de novo pyrimidine and thesalvage pyrimidine pathways of E. coli. The enzymes involved at eachstep are indicated by numbers: 1, carbamoylphosphate synthase; 2,aspartate carbamoyltransferase; 3, dihydroorotase; 4, dihydroorotateoxidase; 5, orotate phosphoribosyltransferase; 6, orotidine 5′-phosphatedecarboxylase; 7, UMP kinase; 8, nucleoside diphosphokinase; 9, CTPsynthetase; 10, ribonucleotide glycosylase; 11, cytosine deaminase; 12,13, uridine phosphorylase; 14, uracil phosphoribosyltransferase; 15,uridine kinase; 16, cytidine deaminase.

[0032]FIG. 15B: The growth characteristics of relevant bacterial strainsillustrating the basis for the selection scheme described in the text.E. coli strains carrying a mutation in codA, the gene encoding CD, areunable to metabolize cytosine. A strain carring a mutation, such aspyrF, in the pyrimidine de novo pathway is dependent on an externalsource of pyrimidines. The presence of both mutations results in astrain that is unable to utilize cytosine as the sole pyrimidine sourceunless the gene encoding CD is provided in trans.

[0033]FIG. 16: Restriction map of the plasmid pEA001. The solid linerepresents sequences of the vector, pBR322, and the stippled linerepresents the cloned insert containing the codA gene. A linearrepresentation of the EcoRI-BamHI insert is shown below the plasmid map.The codA gene is indicated by a solid line with an arrow showing thedirection of transcription. The scale of each figure is located belowthe figure.

[0034]FIG. 17: Restriction map of the plasmid pEA002. The solid linerepresents sequences of the vector, pBR322, and the stippled linerepresents the cloned insert containing the codA gene. A linearrepresentation of the EcoRI-HindIII insert is shown below the plasmidmap. The codA gene is indicated by a solid line with an arrow showingthe direction of transcription. The scale of each figure is locatedbelow the figure.

[0035]FIG. 18: Restriction maps of plasmid DNA inserts and theirphenotypic characteristics and enzymatic activities. The coding regionof codA is indicated at the top. a. Plasmids pEA001-005 are cloned intopBR322, while plasmids pEA006-0014 are cloned into pBS⁺. b. Phenotyperefers to the ability of a plasmid to allow BA101 to utilize cytosine asa sole pyrimidine source. c. Specific activity is defined as nmolcytosine or 5-FC deaminated/mg protein/min. Specific activity wasmeasured spectrophotometrically as a decrease in absorbance at 285 nm ina 1 ml assay mix containing cell extract in 50 mM Tris-HCl, pH 7.3, and0.5 mM cytosine or 5-FC.

[0036]FIG. 19: Restriction map of the plasmid pEA003. The solid linerepresents sequences of the vector, pBR322, and the stippled linerepresents the cloned insert containing the codA gene. A linearrepresentation of the EcoRI-Bg/11 insert is shown below the plasmid map.The codA gene is indicated by a solid line with an arrow showing thedirection of transcription. The scale of the plasmid is located belowthe figure.

[0037]FIG. 20: PAGE analysis of cell extracts prepared from cultures ofBA 101 transformed with various plasmids. Lanes: 1, pBR322; 2, 3,pEA006; 4, pEA005; 5, pEA001; 6, pEA003; 7, pEA004; 8, pEA001; 9,pEA006; 10, pEA009, 11, pEA013; and 12, pEA014. The extracts in lanes1-7 were prepared from cultures grown in minimal medium, while those inlanes 8-12 were prepared from cultures grown in LB. The arrow points tothe CD band, and the molecular weight markers are indicated to the left.

[0038]FIG. 21: Sequence of codA extending from the PstI site to thePvuII site (SEQ ID NO:6). The coding region is translated underneath theDNA sequence with the amino acids verified by protein sequencingunderlined.

[0039]FIG. 22: Growth rates of mixed WiDr and WiDr/CD cells. This graphshows fluorescence units of 96 well microtiter dishes that were platedon Day-1 with 3000 cells/well at the ratios indicated in the legend. Theplates were stained and read on Days 0, 3, 7, and 8.

[0040]FIG. 23: 5-FC dose response of mixed WiDr and WiDr/CD cells. Thisgraph shows fluorescence units of 96 well microtiter dishes that wereplated on Day-1 with 3000 cells/well at the ratios indicated in thelegend. Beginning on Day 3, the cells were dosed with serially diluted5-FC at the concentrations indicated on the x axis. The plates werestained and read on Day 8.

SUMMARY OF THE INVENTION

[0041] Gene therapy involves the stable integration of new genes intotarget cells and the expression of those genes, once they are in place,to alter the phenotype of that particular target cell (for review seeAnderson, W. F. Science 226, 401-409 (1984) and McCormick, D.Biotechnology 3, 689-693, (1985)). Gene therapy may be beneficial forthe treatment of genetic diseases that involve the replacement of onedefective or missing enzyme, such as; hypoxanthine-guaninephosphoribosyl transferase in Lesch-Nyhan disease, purine nucleosidephosphorylase in severe immunodeficiency disease, and adenosinedeaminase in severed combined immunodeficiency disease.

[0042] It has now been found that it is possible to selectively arrestthe growth of, or kill, mammalian carcinoma cells with chemical agentscapable of selective conversion to cytotoxic (causing cell death) orcytostatic (suppressing cell multiplication and growth) metabolites.This is achieved by the construction of a molecular chimaera comprisinga “target tissue-specific” transcriptional regulatory sequence (TRS)that is selectively activated in target cells, such as cancerous cells,and that controls the expression of a heterologous enzyme. Thismolecular chimaera may be manipulated via suitable vectors andincorporated into an infective virion. Upon administration of aninfective virion containing the molecular chimaera to a host (e.g.,mammal or human), the enzyme is selectively expressed in the targetcells. Administration of compounds that are selectively metabolised bythe enzyme into metabolites that are either further metabolised to orare, in fact, cytotoxic or cytostatic agents can then be achieved insitu.

[0043] Molecular chimaeras (recombinant molecules comprised of unnaturalcombinations of genes or sections of genes), and infective virions(complete viral particles capable of infecting appropriate host cells)are well known in the art of molecular biology and are further describedhereinafter.

[0044] The invention is generally applicable and is demonstrated withrespect to the treatment of hepatocellular and colorectal carcinomas.

[0045] As mentioned above the overwhelming percentage of mammals whichhave hepatocellular carcinoma (HCC) die from the primary tumour.However, approximately 90% of HCC patients have overt metastatic diseaseat time of death. These metastases exhibit the typical phenotype of theprimary tumour and will also selectively express the heterologous enzymeand selectively activate administered compounds, as herein defined, tocytotoxic or cytostatic metabolites.

[0046] A number of enzyme prodrug combinations may be used for thispurpose, providing the enzyme is capable of selectively activating theadministered compound either directly or through an intermediate to acytostatic or cytotoxic metabolite. The choice of compound will alsodepend on the enzyme system used, but must be selectively metabolised bythe enzyme either directly or indirectly to a cytotoxic or cytostaticmetabolite. The term heterologous enzyme, as used herein, refers to anenzyme that is derived from or associated with a species which isdifferent from the host to be treated and which will display theappropriate characteristics of the abovementioned selectivity. Inaddition, it will also be appreciated that a heterologous enzyme mayalso refer to an enzyme that is derived from the host to be treated thathas been modified to have unique characteristics unnatural to the host.

[0047] The varicella zoster virus (VZV) encodes a specific thymidinekinase protein. The gene has been cloned, sequenced, and characterised(Davison A. J., Scott J. E. J. Gen. Virol. 67, 1759-1816 (1986)). TheVZV thymidine kinase will, in contrast to the mammalian enzyme,selectively monophosphorylate specific purine arabinosides andsubstituted pyrimidine compounds. It has now been found that certainpurine and pyrimidine analogues of Formulas (I) and (II), particularlythose of Formula I as hereinafter defined, are converted to cytotoxic orcytostatic metabolites in specific mammalian cells that have beengenetically modified to selectively express VZV thymidine kinase. Forexample 9-(β-D-arabinofuranosyl)-6-methoxy-9H-purine is converted to(9-β-1D-arabinofuranosyl)-adenine triphosphate (Ara ATP) which iscytotoxic.

[0048] Other enzyme prodrug combinations include the bacterial (forexample, from Pseudomonas) enzyme carboxypeptidase G2 with the prodrugpara-N-bis-(2-chloroethyl)-aminobenzoyl glutamic acid. Cleavage of theglutamic acid moiety from this compound releases a toxic benzoic acidmustard. Alkaline phosphatase from, for example, calf intestine, willconvert inactive phosphorylated compounds such as etoposide phosphate,doxorubicin phosphate, and mitomycin phosphate to toxic dephosphorylatedmetabolites. Penicillin-V amidase will convert phenoxyacetamidederivatives of doxorubicin and melphalan to toxic metabolites, andcytosine deaminase (for example from E. coli) will convert5-fluorocytosine to toxic 5-fluorouracil.

[0049] The enzyme cytosine deaminase catalyzes the deamination ofcytosine to uracil. Cytosine deaminase is present in microbes and fungibut absent in higher eukaryotes. This enzyme catalyzes the hydrolyticdeamination of cytosine and 5-fluorocytosine (5-FC) to uracil and5-fluorouracil (5-FU), respectively. Since mammalian cells do notexpress significant amounts of cytosine deaminase, they are incapable ofconverting 5-FC to the toxic metabolite 5-FU and therefore5-fluorocytosine is nontoxic to mammalian cells at concentrations whichare effective for antimicrobial activity. 5-Fluorouracil is highly toxicto mammalian cells and is widely used as an anticancer agent.

[0050] In mammalian cells, some genes are ubiquitously expressed. Mostgenes, however, are expressed in a temporal and/or tissue-specificmanner, or are activated in response to extracellular inducers. Forexample, certain genes are actively transcribed only at very precisetimes in ontogeny in specific cell types, or in response to someinducing stimulus. This regulation is mediated in part by theinteraction between transcriptional regulatory sequences (for example,promoter and enhancer regulatory DNA sequences), and sequence-specific,DNA-binding transcriptional protein factors.

[0051] It has now been found that it is possible to alter certainmammalian cells, e.g. liver cells or transformed liver cells, toselectively express a heterologous enzyme as hereinbefore defined, e.g.VZV TK. Colorectal carcinoma cells, metastatic colorectal carcinomacells and hepatic colorectal carcinoma cells can also be altered by thisapproach to selectively express a heterologous enzyme, e.g., cytosinedeaminase. This is achieved by the construction of molecular chimaerasin an expression cassette.

[0052] Expression cassettes themselves are well known in the art ofmolecular biology. Such an expression cassette contains all essentialDNA sequences required for expression of the heterologous enzyme in amammalian cell. For example, a preferred expression cassette willcontain a molecular chimaera containing the coding sequence for VZV TKor cytosine deaminase (CD), an appropriate polyadenylation signal for amammalian gene (i.e., a polyadenylation signal that will function in amammalian cell), and suitable enhancers and promoter sequences in thecorrect orientation.

[0053] Normally, two DNA sequences are required for the complete andefficient transcriptional regulation of genes that encode messenger RNAsin mammalian cells: promoters and enhancers. Promoters are locatedimmediately upstream (5′) from the start site of transcription. Promotersequences are required for accurate and efficient initiation oftranscription. Different gene-specific promoters reveal a common patternof organisation. A typical promoter includes an AT-rich region called aTATA box (which is located approximately 30 base pairs 5′ to thetranscription initiation start site) and one or more upstream promoterelements (UPEs). The UPEs are a principle target for the interactionwith sequence-specific nuclear transcriptional factors. The activity ofpromoter sequences is modulated by other sequences called enhancers. Theenhancer sequence may be a great distance from the promoter in either anupstream (5′) or downstream (3′) position. Hence, enhancers operate inan orientation- and position-independent manner. However, based onsimilar structural organisation and function that may be interchanged,the absolute distinction between promoters and enhancers is somewhatarbitrary. Enhancers increase the rate of transcription from thepromoter sequence. It is predominantly the interaction betweensequence-specific transcriptional factors with the UPE and enhancersequences that enable mammalian cells to achieve tissue-specific geneexpression. The presence of these transcriptional protein factors(tissue-specific, trans-activating factors) bound to the UPE andenhancers (cis-acting, regulatory sequences) enables other components ofthe transcriptional machinery, including RNA polymerase, to initiatetranscription with tissue-specific selectivity and accuracy.

[0054] The selection of the transcriptional regulatory sequence, inparticular the promoter and enhancer sequence will depend on thetargeted cells. Examples include the albumin (ALB) and alpha-fetoprotein(AFP) transcriptional regulatory sequence (for example, the promoter andenhancer) specific for normal hepatocytes and transformed hepatocytes,respectively; the transcriptional regulatory sequence forcarcinoembryonic antigen (CEA) for use in colorectal carcinoma,metastatic colorectal carcinoma, and hepatic colorectal metastases,transformed cells of the gastrointestinal tract, lung, breast and othertissues; the transcriptional regulatory sequence for tyrosinehydroxylase, choline acetyl transferase, or neuron specific enolase foruse in neuroblastomas; the transcriptional regulatory sequence for glialfibro acidic protein for use in gliomas; and the transcriptionalregulatory sequence for insulin for use in tumours of the pancreas.

[0055] Further examples include the transcriptional regulatory sequencespecific for gama-glutamyltranspeptidase for use in certain livertumours and dopa decarboxylase for use in treating certain tumours ofthe lung.

[0056] In addition, the transcriptional regulatory sequences fromcertain oncogenes may be used as these are expressed predominantly incertain tumour types. Good examples of these include the HER-2/neuoncogene regulatory sequence, which is expressed in breast tumours, andthe regulatory sequence specific for the N-myc oncogene forneuroblastomas.

[0057] The ALB and AFP genes exhibit extensive homology with regard tonucleic acid sequence, gene structure, amino acid sequence, and proteinsecondary folding (for review see Ingram R. S., Scott R. W., Tilghman S.M. PNAS 78, 4694-4698 (1981)). These genes are independently butreciprocally expressed in ontogeny. In normal development, ALBtranscription is initiated shortly before birth and continues throughoutadulthood. Transcriptional expression of ALB in the adult is confined tothe liver. AFP is normally expressed in fetal liver, the visceralendoderm of the yolk sac, and the fetal gastrointestinal tract, butdeclines to undetectable levels shortly after birth and is notsignificantly expressed in nonpathogenic or nonregenerating adult liveror in other normal adult tissue. However, AFP transcription in adultliver often increases dramatically in HCC. In addition, AFPtranscription may also be elevated in nonseminomatous and mixedcarcinoma of the testis, in endodermal sinus tumours, in certainteratocarcinomas, and in certain gastrointestinal tumours.Liver-specific expression of AFP and ALB is the result of interactionsof the regulatory sequences of their genes with trans-activatingtranscriptional factors found in nuclear extracts from liver. The AFPand ALB transcriptional regulatory sequences are preferred forgenerating hepatoma-specific or general liver-specific expressionrespectively, of molecularly combined genes because the AFP and ALBgenes are regulated at the transcriptional level and their mRNAs areamong the most abundant polymerase II transcripts in the liver.

[0058] Several mammalian ALB and AFP promoter and enhancer sequenceshave been identified (for review see Pinkert C. A., Ornitz D. M.,Brinster R. L., Palmiter R. D. Genes Dev. 1, 268-276 (1987); Hammer R.E., Krumlauf R., Camper S. A., Brinster R. L. Science 235, 53-58 (1987);Wantanabe K., Saito A., Tamaoki T. The J. of Biol. Chem. 262, 4812-4818(1987)). These sequences enable the selective and specific expression ofgenes in liver hepatocytes (normal and transformed) and hepatomas,respectively.

[0059] For example, as shown in FIG. 1, a mammalian ALB promoter iscontained within a 300-bp fragment 5′ to the transcription initiationstart site of the albumin gene. The sequence contained between 300 bp 5′and 8,500 bp 5′ to the transcription initiation start site of the murinealbumin gene is dispensable for liver-specific expression. However, aliver-specific enhancer sequence is contained in a fragment located from8,500 bp 5′ to 10,400 bp 5′ to the transcription initiation start site(FIG. 1A). If the ALB promoter and enhancer elements are present,liver-specific expression of a heterologous structural gene positionedin a proper 3′ orientation can be achieved (FIG. 1 B). Liver-specificexpression of a 3′ heterologous structural gene positioned in the properorientation to the ALB promoter and enhancer sequences is alsomaintained when the nonessential intervening sequences located between300 bp 5′ and 8,500 bp 5′ to the transcription initiation start site areeliminated (FIG. 1C). The truncation of nonessential sequences isaccomplished by standard molecular biological methodology well known inthe art and results in a molecular chimaera that can be used to directliver-specific expression of VZV TK or any other heterologous enzyme.

[0060] Similar to the regulatory structure of the ALB gene, theregulatory elements of the AFP genes promote tissue-specific expressionof AFP in certain liver pathologies such as HCC (Godbout R., Ingram R.,Tilghman S. M. Mol.Cell.Biol. 6, 477-487 (1986); Hammer R. E., KrumlaufR., Camper S. A., Brinster R. L. Science 235, 53-58 (1987)). Theregulatory elements of a mammalian AFP gene consist of a specific 5′promoter-proximal region (located in some mammalian species between 85and 52 bp 5′ to the gene). This sequence is essential for transcriptionin hepatomas. In addition, there are upstream (5′) regulatory elementswell-defined for the murine AFP gene, which behave as classicalenhancers (Godbout R., Ingram R., Tilghman S. M. Mol.Cel.Biol. 6,477-487 (1986); Hammer R. E., Krumlauf R., Camper S. A., Brinster R. L.Science 235, 53-58 (1987)). These upstream regulatory elements aredesignated elements 1, II, and III and are located between 1,000 to7,600 bp 5′ to the transcription initiation start site for the murineAFP gene. These three enhancer domains are not functionally equivalentat generating tissue-specific expression of AFP. Elements I and II havethe greatest capacity to direct liver-specific expression of AFP. It isimportant to note that the regulatory sequences of the alpha-fetoproteingene advantageously contain the sequences not only for tissue-specifictranscriptional activation but also for repression of expression intissues that should not express AFP. In a similar fashion the regulatoryregions of the human alpha-fetoprotein gene have been characterised(Wantanabe K., Saito M., Tamaoki T. J.Biol.Chem. 262, 4812-4818 (1987)).A structural gene placed in the correct orientation 3′ to the AFPregulatory sequences will enable that structural gene to be selectivelyexpressed in fetal liver, hepatomas, non-seminomatous carcinomas of thetestis, certain teratocarcinomas, certain gastrointestinal tumours andother normal and pathological cells or tissues that specifically expressAFP.

[0061] Carcinoembryonic antigen (CEA) is a tumor-associated marker thatis expressed in a large percentage of primary and metastatic CRC cellsand is widely used as an important diagnostic tool for postoperativesurveillance, chemotherapy efficacy determinations, immunolocalizationand immunotherapy. By placing the expression of the gene encoding CDunder the transcriptional control of the CRC-associated marker gene,CEA, the nontoxic compound, 5-FC, can be metabolically activated to 5-FUselectively in CRC cells (for example, hepatic CRC cells).

[0062] CEA genomic clones were identified and isolated from the humanchromosome 19 genomic library LL19NL01, ATCC number 57766, by standardtechniques described hereinafter.

[0063] The cloned CEA sequences (FIG. 12) comprise CEA enhancers inaddition to the CEA promoter. The CEA enhancers are especiallyadvantagous for high level expression in CEA-positive cells and noexpression in CEA-negative cells.

[0064] Cytosine deaminase clones were identified and isolated frombacteriophage lambda clones by standard techniques describedhereinafter.

[0065] It will be appreciated by those skilled in the art thatnon-dividing normal cells will not have the new genes incorporated bythe infective virions. Therefore, cells which contain ALB but are notactively dividing will not express the heterologous enzyme and willtherefore be unable to metabolise the non-toxic compounds to cytotoxicor cytostatic agents.

[0066] A further advantage of this system is that the generated toxiccompound, 5-fluorouracil, can diffuse out of the cell in which it wasgenerated and kill adjacent tumor cells which did not incorporate theartificial gene for cytosine deaminase.

DETAILED DESCRIPTION OF THE THE INVENTION

[0067] The present invention provides a molecular chimaera comprising atranscriptional regulatory sequence capable of being selectivelyactivated in target tissue or cells, a DNA sequence operatively linkedto the transcriptional regulatory sequence and encoding a heterologousenzyme, the enzyme capable of catalyzing the production of an agentcytotoxic or cytostatic to the target cells.

[0068] Preferably, the target tissue or cells are selected from thegroup consisting of hepatocellular carcinoma, colorectal carcinoma,metastatic colorectal carcinoma, and hepatic colorectal carcinomametastases.

[0069] The present invention further provides a molecular chimaeracomprising a DNA sequence containing the coding sequence of the genethat codes for a heterologous enzyme under the control of atranscriptional regulatory sequence (TRS) in an expression cassette; thetranscriptional regulatory sequence capable of functioning selectivelyin a target tissue or cancer cell, for example one which is capable oftransforming a cancer cell to selectively express an enzyme, forexample, thymidine kinase or cytosine deaminase.

[0070] The present invention further provides in a preferred embodimenta molecular chimaera comprising a transcriptional regulatory sequence,in particular a promoter that is selectively activated in mammaliantarget tissue or cells, which is operatively linked to the codingsequence for the gene encoding varicella zoster virus thymidine kinase(VZV TK) or non-mammalian cytosine deaminase (CD).

[0071] The molecular chimaera comprises a promoter and additionallycomprises an enhancer.

[0072] In particular, the present invention provides a molecularchimaera comprising a DNA sequence of the coding sequence of the genecoding for the heterologous enzyme, which is preferably cytosinedeaminase, additionally including an appropriate polyadenylationsequence (for example see FIG. 2B), which is linked downstream in a 3′position and in the proper orientation to a mammalian targettissue-specific transcriptional regulatory sequence. Most preferably theexpression cassette also contains an enhancer sequence.

[0073] The DNA sequence encoding a heterologous enzyme is additionallyselected from; carboxypeptidase G2; alkaline phosphatase; penicillin-Vamidase; and non-mammalian (e.g., Escherichia coli (E. coli)) cytosinedeaminase (for example see FIG. 12A).

[0074] Preferably non-mammalian cytosine deaminase is selected from thegroup consisting of bacterial, fungal, and yeast cytosine deaminase.

[0075] The promoter and enhancer sequences preferably are selected fromthe transcriptional regulatory sequence for one of albumin (ALB),alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cytomegalovirus(CMV), tryrosine hydroxylase, choline acetyl transferase,neuron-specific enolase, glial fibro acidic protein, insulin orgama-glutamyl-transpeptidase, dopa-decarboxylase, HER-2/neu or N-myconcogene or other suitable genes such as cytomegalovirus (CMV), SV40 orActin. Most preferably the regulatory sequence for ALB or AFP are usedto direct liver-specific or hepatoma-specific expression respectivelyand the regulatory sequence for CEA is used to direct colorectalcarcinoma, metastatic colorectal carcinoma (e.g., hepatic colorectalcarcinoma metastases) specific expression.

[0076] According to the invention, the regulatory sequence for ALB orAFP are also used to direct colorectal carcinoma or metastaticcolorectal carcinoma (e.g., hepatic colorectal carcinoma metastases)specific expression.

[0077] Furthermore, according to the invention, the regulatory sequencefor CEA is also used to direct liver-specific or hepatoma-specificexpression.

[0078] Preferably, the DNA sequence encodes the gene for varicellazoster virus thymidine kinase and is operatively linked to thetranscriptional regulatory sequence for albumin or alpha-fetoprotein.

[0079] Preferably, the DNA sequence encodes the gene for cytosinedeaminase and is operatively linked to the transcriptional regulatorysequence for carcino-embryonic antigen.

[0080] Another aspect of the invention is the genomic CEA sequence asdescribed by FIG. 12A.

[0081] The molecular chimaera of the present invention may be madeutilizing standard recombinant DNA techniques. Thus the coding sequenceand polyadenylation signal of, for example, the VZV thymidine kinase(VZV TK) gene (see FIGS. 2A and 2B) is placed in the proper 3′orientation to the essential ALB or AFP transcriptional regulatoryelements. These molecular chimaeras enable the selective expression ofVZV TK in cells or tissue that normally express ALB or AFP, respectively(FIGS. 3A and 3B). Expression of the VZV TK gene in mammalian liver,hepatomas, certain tumours of the gastrointestinal tract,nonseminomatous carcinomas of the testis, certain teratocarcinomas, andother tumours will enable relatively nontoxic arabinosides andpyrimidines as herein defined to be selectively metabolised to cytotoxicand/or cytostatic metabolites thereof.

[0082] The coding sequence of cytosine deaminase and a polyadenylationsignal (for example see FIGS. 12A and 12B) are placed in the proper 3′orientation to the essential CEA transcriptional regulatory elements.This molecular chimaera enables the selective expression of CD in cellsor tissue that normally express CEA. Expression of the CD gene inmammalian CRC and metastatic CRC (hepatic colorectal carcinomametastases) will enable nontoxic 5-fluorocytosine to be selectivelymetabolised to cytotoxic 5-fluorouracil.

[0083] Accordingly, in a second aspect of the present invention, thereis provided a method of constructing a molecular chimaera comprisinglinking a DNA sequence encoding a heterologous enzyme gene, e.g. VZV TKor CD, to a tissue-specific promoter.

[0084] In particular the present invention provides a method ofconstructing a molecular chimaera as herein defined, the methodcomprising ligating a DNA sequence encoding the coding sequence andpolyadenylation signal of the gene for a heterologous enzyme (e.g., VZVthymidine kinase or non-mammalian CD) to a mammalian tissue-specifictranscriptional regulatory sequence (e.g., promoter sequence andenhancer sequence).

[0085] The VZV thymidine kinase coding sequence and 3′ polyadenylationsignal reside in an approximately 1,381 bp AccI/Nde I restrictionendonuclease fragment (see FIG. 2B).

[0086] Preferably it is the 1,381 bp AccI/Nde I fragment containing theVZV TK coding sequence and polyadenylation signal (FIG. 2B) that isligated to the mammalian tissue-specific promoter and enhancersequences, although it will be appreciated that other DNA fragmentscontaining the VZV TK gene could be used. Moreover, the VZV TKpolyadenylation signal could be replaced with another suitablepolyadenylation signal, such as that from the cytomegalovirus (CMV) orSV40 virus or other mammalian genes.

[0087] Preferably the promoter and enhancer sequences are selected fromthe transcriptional regulatory sequences for one of albumin (ALB),alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), tryrosinehydroxylase, choline acetyl transferase, neuron-specific enolase, glialfibro acidic protein, insulin, gama-glutamyltranspeptidase, dopadecarboxylase, HER-2/neu or N-myc oncogenes or other suitable genes suchas cytomegalovirus (CMV), SV40 or Actin.

[0088] These molecular chimaeras can be delivered to the target tissueor cells by a delivery system. For administration to a host (e.g.,mammal or human), it is necessary to provide an efficient in vivodelivery system that stably incorporates the molecular chimaera into thecells. Known methods utilize techniques of calcium phosphatetransfection, electroporation, microinjection, liposomal transfer,ballistic barrage, DNA viral infection or retroviral infection. For areview of this subject see Biotechniques 6, No.7, (1988).

[0089] The technique of retroviral infection of cells to integrateartificial genes employs retroviral shuttle vectors which are known inthe art (Miller A. D., Buttimore C. Mol. Cell. Biol. 6, 2895-2902(1986)). Essentially, retroviral shuttle vectors (retrovirusescomprising molecular chimaeras used to deliver and stably integrate themolecular chimaera into the genome of the target cell) are generatedusing the DNA form of the retrovirus contained in a plasmid. Theseplasmids also contain sequences necessary for selection and growth inbacteria. Retroviral shuttle vectors are constructed using standardmolecular biology techniques well known in the art. Retroviral shuttlevectors have the parental endogenous retroviral genes (e.g., gag, poland env) removed from the vectors and the DNA sequence of interest isinserted, such as the molecular chimaeras that have been described. Thevectors also contain appropriate retroviral regulatory sequences forviral encapsidation, proviral insertion into the target genome, messagesplicing, termination and polyadenylation. Retroviral shuttle vectorshave been derived from the Moloney murine leukemia virus (Mo-MLV) but itwill be appreciated that other retroviruses can be used such as theclosely related Moloney murine sarcoma virus. Other DNA viruses may alsoprove to be useful as delivery systems. The bovine papilloma virus (BPV)replicates extrachromosomally, so that delivery systems based on BPVhave the advantage that the delivered gene is maintained in anonintegrated manner.

[0090] Thus according to a third aspect of the present invention thereis provided a retroviral shuttle vector comprising the molecularchimaeras as hereinbefore defined. Preferably, the chimaera comprises atranscriptional regulatory sequence which is selectively activated intarget cells and operatively linked to the coding sequence for the geneencoding a heterologous enzyme. The chimaera further comprises a DNAsequence of the coding and polyadenylation sequence of the gene codingfor VZV TK or non-mammalian (e.g., E. coli) cytosine deaminase linked ina 3′ position and in the proper orientation to a transcriptionalregulatory sequence to control expression of the VZV TK gene or CD generespectively. The DNA sequence encoding VZV TK or CD is operativelylinked to a promoter and to a polyadenylation sequence to controlexpression of the VZV TK or CD genes.

[0091] The advantages of a retroviral-mediated gene transfer system arethe high efficiency of the gene delivery to the targeted tissue orcells, sequence specific integration regarding the viral genome (at the5′ and 3′ long terminal repeat (LTR) sequences) and littlerearrangements of delivered DNA compared to other DNA delivery systems.

[0092] Accordingly in a preferred embodiment of the present inventionthere is provided a retroviral shuttle vector comprising a DNA sequencecomprising a 5′ viral LTR sequence, a cis-acting psi-encapsidationsequence, a molecular chimaera as hereinbefore defined and a 3′ viralLTR sequence (FIG. 4A and FIG. 4B).

[0093] In a preferred embodiment, and to help eliminatenon-tissue-specific expression of the molecular chimaera, the molecularchimaera is placed in opposite transcriptional orientation to the 5′retroviral LTR (FIG. 4A and FIG. 4B). In addition, a dominant selectablemarker gene may also be included that is transcriptionally driven fromthe 5′ LTR sequence. Such a dominant selectable marker gene may be thebacterial neomycin-resistance gene NEO (Aminoglycoside 3′phospho-transferase type II), which confers on eukaroytic cellsresistance to the neomycin analogue Geneticin (antibiotic G418 sulphate;registered trademark of GIBCO) (FIG. 4A and 4B). The NEO gene aids inthe selection of packaging cells that contain these sequences (seebelow).

[0094] The retroviral vector used in the examples is based on theMoloney murine leukemia virus but it will be appreciated that othervectors may be used. Vectors containing a NEO gene as a selectablemarker have been described, for example, the N2 vector (Eglitis M. A.,Kantoff P., Gilboa E., Anderson W. F. Science 230, 1395-1398 (1985)).

[0095] A theoretical problem associated with retroviral shuttle vectorsis the potential of retroviral long terminal repeat (LTR) regulatorysequences transcriptionally activating a cellular oncogene at the siteof integration in the host genome. This problem may be diminished bycreating SIN vectors (FIG. 4A). SIN vectors are self-inactivatingvectors that contain a deletion comprising the promoter and enhancerregions in the retroviral LTR. The LTR sequences of SIN vectors do nottranscriptionally activate 5′ or 3′ genomic sequences. Thetranscriptional inactivation of the viral LTR sequences diminishesinsertional activation of adjacent target cell DNA sequences and alsoaids in the selected expression of the delivered molecular chimaera. SINvectors are created by removal of approximately 299 bp in the 3′ viralLTR sequence (Gilboa E., Eglitis P. A., Kantoff P. W., Anderson W. F.Biotechniques 4, 504-512 (1986)).

[0096] Thus preferably the retroviral shuttle vectors of the presentinvention are SIN vectors.

[0097] Since the parental retroviral gag, pol, and env genes have beenremoved from these shuttle vectors, a helper virus system may beutilised to provide the gag, A, and env retroviral gene products intrans to package or encapsidate the retroviral vector into an infectivevirion. This is accomplished by utilising specialised “packaging” celllines, which are capable of generating infectious, synthetic virus yetare deficient in the ability to produce any detectable wild-type virus.In this way the artificial synthetic virus contains a chimaera of thepresent invention packaged into synthetic artificial infectious virionsfree of wild-type helper virus. This is based on the fact that thehelper virus that is stably integrated into the packaging cell containsthe viral structural genes, but is lacking the psi-site, a cis-actingregulatory sequence which must be contained in the viral genomic RNAmolecule for it to be encapsidated into an infectious viral particle.

[0098] Accordingly, in a fourth aspect of the present invention, thereis provided an infective virion comprising a retroviral shuttle vector,as hereinbefore described, said vector being encapsidated within viralproteins to create an artificial infective, replication-defectiveretrovirus.

[0099] Preferably, the retroviral shuttle vector comprises a shuttlevector comprising a molecular chimaera having the transcriptionalregulatory sequence of AFP, ALB, or CEA. In particular, the shuttlevector contains a AFP/VZV TK chimaera, a ALB/VZV TK chimaera or a CEA/CDchimaera. The shuttle vector can further contain a AFP/CD chimaera, aALB/CD chimaera, or a CEA/VZV-TK chimaera.

[0100] In a fifth aspect of the present invention there is provided amethod for producing infective virions of the present invention bydelivering the artificial retroviral shuttle vector comprising amolecular chimaera of the invention, as hereinbefore described, into apackaging cell line.

[0101] The packaging cell line may have stably integrated within it ahelper virus lacking a psi-site and other regulatory sequence, ashereinbefore described, or, alternatively, the packaging cell line maybe engineered so as to contain helper virus structural genes within itsgenome.

[0102] In addition to removal of the psi-site, additional alterationscan be made to the helper virus LTR regulatory sequences to ensure thatthe helper virus is not packaged in virions and is blocked at the levelof reverse transcription and viral integration.

[0103] Alternatively, helper virus structural genes (i.e., gag, pol, andenv) may be individually and independently transferred into thepackaging cell line. Since these viral structural genes are separatedwithin the packaging cell's genome, there is little chance of covertrecombinations generating wild-type virus.

[0104] Accordingly, the present invention also provides for a packagingcell line comprising an infective virion, as described hereinbefore,said virion further comprising a retroviral shuttle vector.

[0105] Accordingly, the present invention provides for a packaging cellline comprising a retroviral shuttle vector as described hereinbefore.

[0106] In addition to retroviral-mediated gene delivery of the chimeric,artificial, therapeutic gene, other gene delivery systems known to thoseskilled in the art can be used in accordance with the present invention.These other gene delivery systems include other viral gene deliverysystems known in the art, such as the adenovirus delivery systems.

[0107] Non-viral delivery systems can be utilized in accordance with thepresent invention as well. For example, liposomal delivery systems candeliver the therapeutic gene to the tumor site via a liposome. Liposomescan be modified to evade metabolism and/or to have distinct targettingmechanisms associated with them. For example, liposomes which haveantibodies incorporated into their structure, such as antibodies to CEA,can have targetting ability to CEA-positive cells. This will increaseboth the selectivity of the present invention as well as it's ability totreat disseminated disease (metastasis).

[0108] Another gene delivery system which can be utilized according tothe present invention is receptor-mediated delivery, wherein the gene ofchoice is incorporated into a ligand which recognizes a specific cellreceptor. This system can also deliver the gene to a specific cell type.Additional modifications can be made to this receptor-mediated deliverysystem, such as incorporation of adenovirus components to the gene sothat the gene is not degraded by the cellular lysosomal compartmentafter internalization by the receptor.

[0109] The present invention further provides an infective virion ashereinbefore described for use in therapy, particularly for use in thetreatment of cancer and more particularly for use in the treatment ofHCC, CRC, metastatic CRC, hepatic CRC metastases, nonseminomatouscarcinoma of the testis, certain teratocarcinomas and certaingastrointestinal tumours.

[0110] The present invention further provides a method of generatingcytosine deaminase in a cell which comprises delivering a molecularchimaera into a cell, said chimaera capable of expressing cytosinedeaminase inside said cell.

[0111] The present invention further provides a method of killing orarresting the growth of cells comprising delivering a molecular chimaerainto said cell, said chimaera expressing a heterologous enzyme (e.g.,cytosine deaminase) in said cells and exposing said cells to an agent(e.g., 5-fluorocytosine) which is converted by said enzyme to an agentwhich is cytotoxic or cytostatic to said cells (e.g., 5-fluorouracil).

[0112] Selective expression of the heterologous enzyme, in particularthe VZV TK gene or CD gene, is accomplished by utilisingtissue-specific, transcriptional regulatory (e.g., enhancer andpromoter) sequences. Selectivity may be additionally improved byselective infection of target tissue or cells, for example, liver cellsor hepatic metastatic colorectal carcinoma cells. The retroviral envgene present in the packaging cell line defines the specificity for hostinfection. The env gene used in constructing the packaging cell line ismodified to encode a ligand for a cell specific binding site to generateartificial, infective virions that selectively infect specific cells,for example, hepatocytes. As an example a retroviral env gene introducedinto the packaging cell may be modified in such a way that theartificial, infective virion's envelope glycoprotein will selectivelyinfect hepatocytes via the specific receptor mediated binding pathwayutilised by the hepatitis B virus (HBV).

[0113] HBV primarily infects hepatocytes via specific receptor mediatedbinding.

[0114] The HBV proteins encoded by the pre-S1 and pre-S2 sequences playa major role in the attachment of HBV to hepatocytes (Hepadna Virusesed. Robinson W., Koike K., Will H. N. Y., A. R. Liss, 189-203, 205-221(1987)). The env gene of the packaging cell is modified to include thehepatocyte binding site of the large S HBV envelope protein. Suchmodifications of the env gene introduced into the packaging cell may beperformed by standard molecular biology techniques well known in theart.

[0115] The infective virion or the packaging cell line according to theinvention may be formulated by techniques well known in the art and maybe presented as a formulation (composition) with a pharmaceuticallyacceptable carrier therefor. Pharmaceutically acceptable carriers, inthis instance physiologic aqueous solutions, may comprise liquid mediumsuitable for use as vehicles to introduce the infective virion into ahost. An example of such a carrier is saline. The infective virion orpackaging cell line may be a solution or suspension in such a vehicle.Stabilizers and antioxidants and/or other excipients may also be presentin such pharmaceutical formulations (compositions), which may beadministered to a mammal by any conventional method (e.g., oral orparenteral routes). In particular, the infective virion may beadministered by intra-venous or intra-arterial infusion. In the case oftreating HCC or hepatic metastatic CRC, intra-hepatic arterial infusionmay be advantageous. The packaging cell line can be administereddirectly to the tumor or near the tumor and thereby produce infectivevirions directly at or near the tumor site.

[0116] Accordingly, the invention provides a pharmaceutical formulation(composition) comprising an infective virion or packaging cell lineaccording to the invention in admixture with a pharmaceuticallyacceptable carrier.

[0117] Additionally, the present invention provides methods of makingpharmaceutical formulations (compositions), as herein described,comprising mixing an artificial infective virion, containing a molecularchimaera according to the invention as described hereinbefore, with apharmaceutically acceptable carrier.

[0118] The present invention also provides methods of makingpharmaceutical formulations (compositions), as herein described,comprising mixing a packaging cell line, containing an infective virionaccording to the invention as described hereinbefore, with apharmaceutically acceptable carrier.

[0119] Although any suitable compound that can be selectively convertedto a cytotoxic or cytotostatic metabolite by the enzyme may be utilised,the present invention further provides the use of compounds of Formulas(I) or (II) in the manufacture of a medicament for use in treatingcancers capable of expressing VZV TK. In particular for use in treatinghepatocellular carcinoma (HCC).

[0120] 6-Substituted purine arabinoside compounds of Formula (I), theirsalts, esters, and physiologically functional equivalents thereof areshown hereinbelow:

[0121] wherein

[0122] R₁ is halo, C₁₋₅ alkoxy, halogen-substituted C₁₋₅ alkoxy; anamino group which is mono- or di-substituted by C₁₋₅ alkyl, C₁₋₅ alkylsubstituted by one or more fluorine atoms, C₃₋₆ cycloalkyl, or anitrogen-containing heterocycle containing 4-7 carbon atoms andoptionally a double bond; and R₂ is hydrogen, halo, or amino; arepurine-arabino nucleosides which have been reported to have potentactivity against human virus infections particularly those caused byvaricella zoster virus (VZV) and cytomegalovirus (CMV) (European patentapplication number 88304813.4 filed May 27, 1988 and published Dec. 7,1988(Bulletin 88/49) under number 0294114) which is herein incorporatedby reference in its entirety.

[0123] Certain substituted purine-arabino nucleosides, in particular9-β-D-arabinofuranosyl-6-methoxy-9-H-purine,9-β-D-arabinofuranosyl-6-pyrrolidino-9-H-purine,9-β-D-arabinofuranosyl-6-methylamino-9-H-purine, and9-β-D-arabinofuranosyl-6-dimethylamino-9-H-purine, have previously beendescribed in J. Org. Chem., 27, 3274-3279(1962); Cancer Treatment Rep.,60(10), 1567-1584(1976); Tetrahedron, 40(4), 709-713(1984); Canada J.Biochem., 43(1), 1-15(1965); J. Med. Chem., 12, 498-504(1969); J. Biol.Chem., 251(13), 4055-4061(1976); Ann. N.Y. Acad. Sci., 284, 81-90(1977)which are herein incorporated by reference in their entirety.

[0124] The following compounds of Formula (I) are preferred compounds tobe used in accordance with the present invention;

[0125] 9-β-D-arabinofuranosyl-6-methylamino-9-H-purine.

[0126] 9-β-D-arabinofuranosyl-6-dimethylamino-9-H-purine.

[0127] 9-β-D-arabinofuranosyl-6-methoxy-9-H-purine.

[0128] 9-β-D-arabinofuranosyl-6-ethoxy-9-H-purine.

[0129] 9-β-D-arabinofuranosyl-6-iodo-9-H-purine.

[0130] 9-β-D-arabinofuranosyl-2-amino-6-iodopurine.

[0131] 9-β-D-arabinofuranosyl-6-pyrrolid ino-9-H-purine.

[0132] 9-β-D-arabinofuranosyl-2-chloro-6-methylamino-9-H-purine.

[0133] 9-β-D-arabinofuranosyl-6-cyclopropylamino-9-H-purine.

[0134] 9-β-D-arabinofuranosyl-6-ethylmethylamino-9-H-purine.

[0135] 9-β-D-arabinofuranosyl-2-amino-6-methoxy-9-H-purine.

[0136] 9-β-D-arabinofuranosyl-6-n-propoxy-9-H-purine.

[0137] Of the above compounds,9-β-D-arabinofuranosyl-6-methoxy-9-H-purine is especially preferred.

[0138] The compounds of Formula (I) to be used in accordance with thepresent invention may be prepared by methods known in the art for thepreparation of the same or similar compounds.

[0139] 5-Substituted pyrimidine nucleoside compounds of Formula (II),their salts, esters, and physiologically functional equivalents thereofare shown hereinbelow:

[0140] wherein X represents a vinylene or ethynylene group; R¹represents an oxo or imino group; R² represents a hydrogen atom, C₁₋₂alkyl, C₃₋₄ branched or cycloalkyl group (e.g., isopropyl orcyclopropyl); R³ represents a hydrogen atom or an acyl (e.g., C₁₋₄alkanoyl or benzoyl) group optionally substituted, for example, by oneor more halogen, alkyl, hydroxy, or alkoxy substituents; and R⁴represents a hydrogen atom or a hydroxy group.

[0141] These pyrimidine nucleosides which are characterized by thepresence of an unsaturated grouping in the 5-position, have previouslybeen shown to have anti-VZV activity as well as a relatively low levelof toxicity (European patent application number 87310951.6 filed Dec.14, 1987 and published Jun. 22, 1988 (Bulletin 88/25) under number0272065) which is herein incorporated by reference in its entirety.

[0142] Certain 5-substituted nucleosides, in particular2′-deoxy-5-(1-propynyl)uridine, 2′-deoxy-5-ethynylcytidine,1-(β-D-arabinofuranosyl)-5-propynyluracil,1-(β-D-arabinofuranosyl)-5-ethynylcytosine have previously beendescribed in J. Med. Chem., 26(5), 661-666(1983); J. Med. Chem., 26(9),1252-1257(1983); Antimicrobial Agents Chemother., 17(6),1030-1031(1980); Nucleic Acids Symp. Ser., 9, 103-106(1981); Biochem.Pharmacol., 32(4), 726-729(1983) which are herein incorporated byreference in their entirety.

[0143] It will be appreciated that when R³ is not an acyl group, thecompounds of Formula (II) may exist in their tautomeric form.

[0144] The following compounds of Formula (II) are preferred compoundsto be used in accordance with the present invention;

[0145] 2′-Deoxy-5-(1-propynyl)uridine.

[0146] 2′-Deoxy-5-ethynylcytidine.

[0147] 3-N-Benzoyl-2′-deoxy-5-ethynyluridine.

[0148] 1-(β-D-Arabinofuranosyl)-5-ethynyluracil.

[0149] 2′-Deoxy-5-(1-propynyl)cytidine.

[0150] 1-(β-D-Arabinofuranosyl)-5-propynylcytosine.

[0151] 3-N-Benzoyl-2′-deoxy-5-propynyluridine.

[0152] 1-(β-D-Arabinofuranosyl)-5-propynyluracil.

[0153] 1-(β-D-Arabinofuranosyl)-5-ethynylcytosine.

[0154] 1-(β-D-Arabinofuranosyl)-3-N-benzoyl-5-propynyluracil.

[0155] 1-(β-β-D-Arabinofuranosyl)-3-N-benzoyl-5-ethynyluracil.

[0156] 3-N-Benzoyl-2′-deoxy-5-vinyluridine.

[0157] 1-(β-D-Arabinofuranosyl)-3-N-benzoyl-5-vinyluracil.

[0158] A particularly preferred compound of Formula (II) is1-(β-D-arabinofuranosyl)-5-propynyluracil.

[0159] The compounds of Formula (II) to be used according to theinvention may be prepared by any of the methods known in the art for thepreparation of the same or similar compounds (e.g., see Robins M. J.,and Barr, P. J., J. Org. Chem., 43, 1854-1862(1983) which is hereinincorporated by reference in its entirety).

[0160] The abovementioned purine arabinosides and pyrimidine nucleosidesalso include the pharmaceutically acceptable derivatives of suchcompounds, i.e., any pharmaceutically acceptable salt, ester, or salt ofsuch ester, or any other compound which, upon administration to a humansubject, is capable of providing (directly or indirectly) the activemetabolite or residue thereof. Preferably, the compound is orallyactive.

[0161] The pharmaceutically acceptable esters of the above compounds ofFormula (I) are particularly preferred since they are capable ofproviding high levels of the parent compound in the plasma of a subjectafter oral administration. Particularly preferred derivatives ofcompounds of Formula (I) include mono-, di-, or tri-esters of thearabino-sugar residue substituted at the 2′-, 3′-, and 5′-positions ofsaid residue.

[0162] Such preferred esters include carboxylic acid esters in which thenon-carbonyl moiety of the ester grouping is selected from straight orbranched chain alkyl (e.g., n-propyl, t-butyl, n-butyl), alkoxyalkyl(e.g., methoxymethyl), aralkyl (e.g., benzyl), aryloxyalkyl (e.g.,phenoxymethyl), aryl (e.g., phenyl) optionally substituted by halogen,C₁₋₄ alkyl or C₁₋₄ alkoxy, nitro or amino; sulfonate esters such asalkylsulfonyl, or arylsulfonyl (e.g., methanesulfonyl or tosylsulfonyl);amino acid esters (e.g., L-valyl); and mono-, di-, or tri-phosphateesters. Pharmaceutically acceptable salts of these esters includesodium, potassium, NR₄ ⁺ where R═H or C₁₋₆ alkyl, and acid additionsalts. In the above ester groups, the alkyl groups (including those inalkoxy groupings) contain 1 to 12 carbon atoms and the aryl groups arepreferably phenyl.

[0163] The following esters and ethers are preferred compounds to beused in accordance with the present invention:

[0164] 9-(5-O-Benzoyl-β-D-arabinofuranosyl)-6-methoxy-9-H-purine.

[0165]6-Methoxy-9-[5-O-(4-methylphenylsulfonyl)-Aβ-D-arabinofuranosyl]-9-H-purine.

[0166] 6-Methoxy-9-(5-O-methylsulfonyl-β-D-arabinofuranosyl)-9-H-purine.

[0167]9-(5-O-(4-Methylbenzoyl)-β-D-arabinofuranosyl)-6-methoxy-9-H-purine.

[0168]9-(5-O-(4-Chlorobenzoyl)-β-D-arabinofuranosyl)-6-methoxy-9-H-purine.

[0169]9-(5-O-(4-Methoxybenzoyl)-β-D-arabinofuranosyl)-6-methoxy-9-H-purine.

[0170] 6-Methoxy-9-(5-O-phenylacetyl-β-D-arabinofuranosyl)-9-H-purine.

[0171]6-Methoxy-9-(5-O-phenyloxyacetyl-β-D-arabinofuranosyl)-9-H-purine.

[0172] 6-Methoxy-9-(5-O-methoxyacetyl-β-D-arabinofuranosyl)-9-H-purine.

[0173]9-(5-O-(4-Nitrobenzoyl)-β-D-arabinofuranosyl)-6-methoxy-9-H-purine.

[0174] 6-Methoxy-9-(5-O-pentanoyl-β-D-arabinofuranosyl)-9-H-purine.

[0175]9-[5-O-(4-Aminobenzoyl)-β-D-arabinofuranosyl]-6-methoxy-9-H-purine.

[0176] 6-Methoxy-9-(5-O-propionyi-β-D-arabinofuranosyl)-9-H-purine.

[0177] 9-(5-β-Butanoyl-β-D-arabinofuranosyl)-6-methoxy-9-H-purine.

[0178]9-[5-O-(2,2-Dimethylpropionyl)-β-D-arabinofuranosyl]-6-methoxy-9-H-purine.

[0179] 9-(5-O-Acetyl-β-D-arabinofuranosyl)-6-methoxy-9-H-purine.

[0180]6-Methoxy-9-[5-O-(2-methypropionyl)-β-D-arabinofuranosyl]-9-H-purine.

[0181]6-Methoxy-9-[2-O-(2,2-dimethylpropionyl)-β-D-arabinofuranosyl]-9-H-purine.

[0182] 6-Methoxy-9-[(2,3,5-tri-O-acetyl)-D-arabinofuranosyl]-9-H-purine.

[0183] 6-Methoxy-9-(2-O-pentanoyl-β-D-arabinofuranosyl)-9-H-purine.

[0184] 9-(2-O-Butanoyl-β-D-arabinofuranosyl)-6-methoxy-9-H-purine.

[0185]6-Methoxy-9-[2-O-(2-methylpropionyl)-β-D-arabino-furanosyl]-9-H-purine.

[0186] 9-(3-O-Benzoyi-β-D-arabinofuranosyl)-6-methoxy-9-H-purine.

[0187] 9-(2,3-Anhydro-β-D-Iyxofuranosyl)-6-methoxy-9-H-purine.

[0188] 6-Methoxy-9-[(2-O-(4-methoxybenzoyl))-β-D-arabinofuranosyl]-9-H-purine.

[0189]6-Methoxy-9-[(2-O-(4-methylbenzoyl))-β-D-arabinofuranosyl]-9-H-purine.

[0190]9-[2-O-(4-Chlorobenzoyl)-β-D-arabinofuranosyl]-6-methoxy-9-H-purine.

[0191]6-Methoxy-9-[3,5-O-(1,1,3,3-tetraisopropyl-1,3-disiloxan-1,3-diyl)-β-D-arabinofuranosyl]-9-H-purine.

[0192]6-Methoxy-9-[2-O-(2-aminobenzoyl)-β-D-arabinofuranosyl]-9-H-purine.

[0193]6-Methoxy-9-[2-(4-methylbenzoyl)-3,5-O-(1,1,3,3-tetra-isopropyidisiloxan-1,3-diyl)-β-D-arabinofuranosyl]-9-H-purine.

[0194]6-Methoxy-9-[2-(4-methoxybenzoyl)-3,5-O-(1,1,3,3-tetra-isopropyidisiloxan-1,3-diyl)-β-D-arabinofuranosyl]-9-H-purine.

[0195]9-[2-(4-Chlorobenzoyl)-3,5-O-(1,1,3,3-tetra-isopropyldisiloxan-1,3-diyl)-β-D-arabinofuranosyl]-6-methoxy-9-H-purine.

[0196] 5′-Monophosphate ester of9-β-D-arabinofuranosyl-6-dimethylamine-9H-purine.

[0197] 6-Methoxypurine arabinoside 5′-monophosphate.

[0198] 6-Methoxypurine arabinoside 5′-triphosphate.

[0199] 6-Dimethylamino-9-[(2-O-valeryl)-β-D-arabinosyl]-9H-purine.

[0200] 6-Dimethylamino-9-(2,3,5-triacetyl-β-D-arabinosyl)-9H-purine.

[0201] Physiologically acceptable salts and esters of compounds ofFormula (I) to be used according to the present invention may beprepared in conventional manner. For example, esters may be prepared byesterification of the parent compound with an appropriate acyl halide oranhydride. Alternatively, the esters may be prepared by displacing theappropriate leaving group (e.g., halide) with an appropriate carboxylicacid or by opening an appropriate anhydro nucleoside of the parentcompound with an appropriate carboxylic acid or salt thereof.

[0202] Also pharmaceutically acceptable salts and esters of compounds ofFormula (II) especially the diacetate of 2′-deoxy-5-ethynylcytidine,namely 2′-deoxy-3′,5′-di-O-acetyl-5-ethynylcytidine, may be used inaccordance with the present invention.

[0203] Although any suitable compound that can be selectively convertedto a cytotoxic or cytotostatic metabolite by the enzyme cytosinedeaminase may be utilised, the present invention further provides theuse of 5-fluorocytosine in the manufacture of a medicament for use intreating cancers capable of expressing cytosine deaminase. In particularfor use in treating hepatocellular carcinoma (HCC), colorectal carcinoma(CRC), metastatic colorectal carcinoma, or hepatic CRC metastases.

[0204] Any agent that can potentiate the antitumor effects of 5-FU canalso potentiate the antitumor effects of 5-fluorocytosine (5-FC) since,when used according to the present invention, 5-FC is selectivelyconverted to 5-FU. According to another aspect of the present invention,agents such as leucovorin and levemisol, which can potentiate theantitumor effects of 5-FU, can also be used in combination with 5-FCwhen 5-FC is used according to the present invention. Other agents whichcan potentiate the antitumor effects of 5-FU are agents which block themetabolism 5-FU. Examples of such agents are 5-substituted uracilderivitives, for example, 5-ethynyluracil and 5-bromvinyluracil(PCT/GB91/01650(WO 92/04901); Cancer Research 46, 1094, 1986 which areincorporated herein by reference in their entirety). Therefore, afurther aspect of the present invention is the use of an agent which canpotentiate the antitumor effects of 5-FU, for example, a 5-substituteduracil dervitive such as 5-ethynyluracil or 5-bromvinyluracil incombination with 5-FC when 5-FC is used according to the presentinvention. The present invention further includes the use of agentswhich are metabolised in vivo to the corresponding 5-substituted uracilderivatives described hereinbefore (see Biochemical Pharmacology 38,2885, 1989 which is incorporated herein by reference in its entirety) incombination with 5-FC when 5-FC is used according to the presentinvention.

[0205] 5-Fluorocytosine is readily available (e.g., United StatesBiochemical, Sigma) and well known in the art. Leucovorin and levemisolare also readily available and well known in the art.

[0206] Two significant advantages of the enzyme/prodrug combination ofcytosine deaminase/5-fluorocytosine and further aspects of the inventionare the following:

[0207] 1. The metabolic conversion of 5-fluorocytosine (5-FC) bycytosine deaminase produces 5-fluorouracil (5-FU). 5-FU is the drug ofchoice in the treatment of many different types of cancers, such ascolorectal carcinoma.

[0208] 2. The 5-FU that is selectively produced in one cancer cell candiffuse out of that cell and be taken up by both non-facilitateddiffusion and facilitated diffusion into adjacent cells. This produces aneighboring cell killing effect. This neighbor cell killing effectalleviates the necessity for delivery of the therapeutic molecularchimera to every tumor cell. Rather, delivery of the molecular chimerato a certain percentage of tumor cells can produce the completeeradication of all tumor cells.

[0209] The amounts and precise regimen in treating a mammal, will ofcourse be the responsibility of the attendant physician, and will dependon a number of factors including the type and severity of the conditionto be treated. However, for HCC or hepatic metastatic CRC, anintrahepatic arterial infusion of the artificial infective virion at atiter of between 2×10⁵ and 2×10⁷ colony forming units per mL (CFU/mL)infective virions is suitable for a typical tumour. Total amount ofvirions infused will be dependent on tumour size and are preferablygiven in divided doses.

[0210] Likewise, the packaging cell line is administered directly to atumor in an amount of between 2×10⁵ and 2×10⁷ cells. Total amount ofpackaging cell line infused will be dependent on tumour size and ispreferably given in divided doses.

[0211] Prodrug treatment—Subsequent to infection with the infectivevirion, compounds according to the invention, which are described byFormulas (I) and (II), are administered that specifically require VZV TKactivity for the critical phosphorylation step in anabolism to generatecytotoxic or cytostatic metabolites. These prodrug compounds, which aresubsequently converted to cytotoxic or cytostatic metabolites in thetarget cells, are preferably purine arabinosides or pyrimidinenucleosides. Most preferably 9-β-D-arabinofuranosyl-6-methoxy-9H-purineand 1-(β-D-arabinofuranosyl)-5-propyniuracil. Likewise, certain cytosinecompounds (prodrugs of 5-FU) are converted by cytosine deaminase tocytoxic or cytostatic metabolites (e.g., 5-fluorocytosine is convertedto 5-fluorouracil) in target cells. The abovementioned prodrug compoundsare administered to the host (e.g., mammal or human) between six hoursand ten days, preferably between one and five days, after administrationof the infective virion.

[0212] The dose of compound, as described by Formulas (I) and (II), tobe given will advantageously be in the range 0.1 to 250 mg per Kgm bodyweight of recipient per day, preferably 0.1 to 100 mg per Kgm bodyweightof recipient per day, more preferably 1 to 40 mg per Kgm bodyweight ofrecipient per day, and most preferably 15-40 mg per Kgm body weight ofrecipient per day.

[0213] The dose of 5-fluorocytosine to be given will advantageously bein the range 10 to 500 mg per Kgm body weight of recipient per day,preferably 50 to 500 mg per Kgm bodyweight of recipient per day, morepreferably 50 to 250 mg per Kgm bodyweight of recipient per day, andmost preferably 50 to 150 mg per Kgm body weight of recipient per day.The mode of administration of 5-FC in humans are well known to thoseskilled in the art. Oral administration and/or constant intravenousinfusion of 5-FC are anticipated by the instant invention to bepreferable.

[0214] The doses and mode of administration of leucovorin and levemisolto be used in accordance with the present invention are well known orreadily determined by those clinicians skilled in the art of oncology.

[0215] The dose and mode of administration of the 5-substituted uracilderivitives can be determined by the skilled oncologist. Preferably,these derivatives are given by intravenous injection or orally at a doseof between 0.01 to 50 mg per kilogram body weight of the recipient perday, particularly 0.01 to 10 mg per kilogram body weight per day, andmore preferably 0.01 to 0.4 mg per kilogram bodyweight per day dependingon the deriviative used. An alternative preferred administration regimeis 0.5 to 10 mg per kilogram body weight of recipient once per week.

[0216] The invention also provides a method of treating a host (e.g.,mammal or human) in need of anticancer treatment which comprisesadministering to the host, a molecular chimaera capable of beingselectively activated in the cells of the host to express an enzyme, andsubsequently administering an agent which is converted in the cells bythe enzyme to an agent which is cytotoxic or cytostatic to the cells.Preferably the molecular chimaera expresses the enzyme CD and the agentwhich is converted by the enzyme is 5-FC.

[0217] The invention also comprises a method of killing cells in vitrowhich comprises administering to the cells, a molecular chimaera capableof being selectively activated in the cells to express an enzyme, andsubsequently administering an agent which is converted in the cells bythe enzyme to an agent which is cytotoxic or cytostatic to the cells.Preferably the molecular chimaera expresses the enzyme CD and the agentwhich is converted by the enzyme is 5-FC.

[0218] The invention further provides a method of treating a host inneed of anticancer treatment comprising administering to the host aninfective virion or a packaging cell line as described hereinbefore.Namely, the packaging cell line comprises an infective virionencapsidating a retroviral shuttle vector comprising a molecularchimaera, the chimaera comprising a transcriptional regulatory sequencewhich is selectively activated in cells of the host and operativelylinked to a gene encoding a heterologous enzyme; in an amount sufficientto transform the cells so as to express the enzyme, and subsequentlyadministering to the host an amount of a compound which is selectivelymetabolised in the cells by the enzyme to a cytotoxic or cytostaticmetabolite.

[0219] The following examples serve to illustrate the present inventionbut should not be construed as a limitation thereof.

EXAMPLE 1 Construction of Transcriptional Regulatory Sequence ofAlbumin/VZV Thymidine Kinase Molecular Chimaera

[0220] An approximately 1,381 bp Acc I/Nde 1 DNA fragment (allrestriction enzymes obtained from either Bethesda Research Laboratories,Gaithersburg Md., USA; New England Biolabs, MA, USA; or Promega,Madison, Wis., USA; all enzymatic reactions performed as specified bythe supplier) containing the coding sequence and polyadenylation signalof the VZV TK gene was purified by electroelution using an elutrapelectrophoresis chamber (by Schleicher and Schuell, Keene, N.H., USA)from a restriction endonuclease digestion of an approximately 4,896 bpplasmid, designated 22TK, containing an approximately 2,200 bpEcoRI/BamHI fragment of the VZV TK genome (Sawyer M. H., Ostrove J. M.,Felser J. M. Virology 149 1-9 (1986); supplied by J. Ostrove, NIH,Bethesda, Md.). The purified DNA fragment contains the entire VZV TKcoding sequence and polyadenylation signal, but does not include any VZVTK promotional elements (see FIGS. 2A and 2B)(SEQ ID NO:1).

[0221] The 5′ overhanging ends of the purified 1,381 bp AccI/Nde I VZVTK fragment were made blunt by treatment with the Klenow fragment of E.coli DNA polymerase I (Bethesda Research Laboratories, Gaithersburg,Md., USA) and deoxynucleotide triphosphates (dNTPs).

[0222] An approximately 5,249 bp plasmid, designated 2335A-1, containingapproximately 2,300 bp of a ALB E/P sequence was obtained from RichardPalmiter (University of Washington, Seatle, Wash. USA). This constructcontains sequences necessary for liver-specific expression but lacksnonessential intervening sequences. A unique BamH I restrictionendonuclease recognition site is present at +23 relative to the start oftranscription. 2335A-1 was digested with the restriction endonucleaseBamH I and the 5′ overhanging ends were made blunt by treatment withKlenow and dNTPs as described above.

[0223] The ALB E/P VZV TK chimaera was constructed by ligating the bluntended AccI-NdeI fragment containing the VZV TK coding andpolyadenylation sequences into the blunt ended BamH I site of 2335A-1creating pCR73 using T4 DNA ligase (Bethesda Research Laboratories,Gaithersburg, Md.) (FIG. 5). Similar to all ligations described, theorientation of the ligated fragments was determined by restrictionendonuclease digestions by methods well known in the art. Similar to allplasmids described in all examples, pCR73 contains essential sequencesrequired for replication in bacteria and suitable for amplification bymethods well known in the art. The ALB E/P VZV TK chimaera was purifiedby electroelution from pCR73 as an approximately 3880 bp SstI/KpnIrestriction endonuclease fragment (FIG. 5). The 3′ overhanging ends weremade blunt by treatment with T4 DNA polymerase and dNTPs. This SstI/KpnIblunt ended restriction fragment was subsequently introduced into aMoloney murine leukemia virus retroviral shuttle vector system (seebelow, Example 3).

[0224] pCR73 was deposited at the American Type Culture Collection,Rockville, Md. USA (ATTC) on 18th August 1989 under the Budapest Treatywith Accession No. ATCC68077.

EXAMPLE 2 Construction of Transcriptional Regulatory Sequence ofAlpha-fetoprotein/VZV Thymidine Kinase Molecular Chimaera

[0225] The VZV thymidine kinase coding sequence and polyadenylation sitewas isolated as an approximately 3,300 bp BamHI-XmnI restrictionendonuclease fragment of pCR73 (FIG. 6). The 5′ overhanging end of theBamHI restriction endonuclease site was made blunt by treatment withKlenow and dNTPs. This DNA fragment contains the complete codingsequence and the polyadenylation site of the VZV TK gene but does notcontain any enhancer or promoter sequences. An approximately 9,996 bpplasmid, pAF5.1-CAT, containing an approximately 5,100 bp of human AFP5′ flanking DNA was obtained from T. Tamaoki, Univ. of Calgary, Canada.A DNA fragment spanning from approximately -5.1 kb to +29 of the humanAFP gene was isolated from pAF5.1-CAT by digestion with XmnI and partialdigestion with HindIII. This XmnI/HindII fragment was ligated to theBamHI/XmnI VZV TK fragment using T4 DNA ligase to form pCR77 (FIG. 6).The AFP E/P VZV TK chimera was purified by electroelution from pCR77 asan approximately 6,699 bp Aat II /PstI restriction endonucleasefragment. This fragment was then treated with T4 DNA polymerase anddNTPs as Example 1 to produce a blunt end restriction fragment.

[0226] pCR77 was deposited at the ATCC on Aug. 18, 1989, under theBudapest Treaty with Accession No. ATCC68079.

EXAMPLE 3 Construction of a Retroviral Shuttle Vector ConstructContaining the Molecular Chimaera of Example 1

[0227] The retroviral shuttle vector, pCR74, containing the ALB E/P VZVTK chimaera was constructed by ligating the purified SstI/KpnI bluntended fragment of pCR73 into a Moloney murine leukemia retroviral vectordesignated N2 (XM5) (Eglitis M. A., Kantoff P., Gilboa E., Anderson W.F. Science 230, 1395-1398 (1985)) obtained from S. Karrlson, NIH,Bethesda, Md., USA. N2 (XM5) was digested with the restrictionendonuclease XhoI and the 5′ overhanging ends were made blunt bytreatment with both Kienow and dNTPs prior to the ligation to the pCR73Sstl/Kpnl fragment using T4 DNA ligase (FIG. 5).

[0228] The retroviral shuttle vector pCR74 containing the ALB E/P VZV TKchimaera has been characterised by restriction endonuclease mapping andDNA sequencing to confirm the primary sequence. The sequence flankingthe junction of the ALB E/P to the VZV TK sequences is shown in FIG. 7(SEQ ID NO:2).

[0229] pCR74 was deposited at the ATCC on Aug. 18, 1989 under theBudapest Treaty with Accession No. ATCC68078.

EXAMPLE 4 Construction of a Retroviral Shuttle Vector ConstructContaining the Molecular Chimaera of Example 2

[0230] The retroviral shuttle vector pCR78 (FIG. 6) was constructed byligating a purified AatII/PstI fragment of pCR77 containing the AFP EIPVZV TK chimaera into N2(XM5), which was digested with XhoI and madeblunt ended with T4 DNA polymerase and dNTPs as described in Example 3.The retroviral shuttle vector pCR78 containing the AFP E/P VZV TKchimaera has been characterised by restriction endonuclease mapping andDNA sequencing to confirm the primary sequence. The sequence flankingthe junction of AFP E/P to the VZV TK sequence is shown in FIG. 8 (SEQID NO:3).

[0231] pCR78 was deposited at the ATCC on Aug. 18, 1989 under theBudapest Treaty with Accession No. ATCC68080.

EXAMPLE 5 Virus Production

[0232] The packaging cell line called PA317 obtained from ATCC, (ATCCCRL 9078), which has been previously described, has three alterationscontained within the 5′ LTR, psi regulatory sequence, and 3′ LTR (MillerA. D., Buttimore C. Mol. Cell. Biol. 6 2895-2902 (1986)). The artificialretroviral constructs described in Examples 3 and 4 were placed into thepackaging cell line by electroporation or infection. Forelectroporation, 20 ug of linearized plasmid DNA was electroporated into2 million PA317 cells in phosphate buffered sucrose using 280 volts, 25microfarads of capacitance in a total volume of 0.8 mLs. There wasobtained at least about 150 G418 resistant colonies/20 ug plasmid DNA/2million PA317 cells. For infection, 20 ug of linearized plasmid DNA waselectroporated into 2 million ecotropic packaging cells, such as Psi 2cells. Two days later, the culture supernatant was used to infect theamphotropic packaging cell line PA317 and G418 resistant coloniesisolated. For both electroporation and infection techniques, G418resistant colonies were single cell cloned by the limiting dilutionmethod, analysed by Southern blots, and titered in NIH 3T3 cells (ATCC)to identify the highest producer of full-length virus. For PA317 cellscontaining pCR74, 17 single-cell clones were isolated and DNA wasextracted from 10 of these clones. Extensive Southern blot analysisusing different restriction endonuclease enzymes and NEO and VZV TKsequences as radioactive hybridization probes was performed on these 10DNA samples. Out of the 10 clones analysed, two showed no evidence oftruncation and are considered full length. For PA317 cells containingpCR78, 29 single-cell clones were isolated and DNA was obtained from 25clones. Extensive Southern blot analysis using different restrictionendonuclease enzymes and AFP, NEO, and VZV TK sequences as radioactivehybridisation probes was performed on these 25 samples. Out of the 25clones analysed, 5 showed no evidence of truncation and are consideredfull length. Each packaging cell line containing a full length viralsequence was titered in NIH 3T3 cells, which were thymidine kinaseminus/minus, using standard techniques.

EXAMPLE 6 Infection of Human Hepatoma Cell Lines (Positive Controls)with Full Length Infective Virions of Example 5 Containing ALB/VZV TK orAFP/VZV TK with Subsequent Measurements of VZV TK Activity, Ara-ATPProduction and Drug Sensitivity

[0233] The replication-defective, full-length, artificial retrovirusescontaining the ALB/VZV TK chimaera or AFP/VZV TK chimaera were used toinfect human hepatoma cell lines called HepG2 (ATCC HB 8065) andHuH7(provided by B. Mason, Fox Chase Cancer Center, Philadelphia, Pa.).Following infection and selection on 1 mg Geneticin/mL, the cells wereassayed for VZV TK activity (Geneticin(antibiotic G418 sulphate) is aregistered trademark of GIBCO). In addition, HepG2 cells were incubatedin the presence of (³H)-labeled9-β-D-arabinofuranosyl-6-methoxy-9H-purine (designated as araM in thefollowing tables and figures) with subsequent measurement of ara-ATPformation. Finally, HepG2 and HuH7 cells were cultured in the presenceof the abovementioned compound and the IC₅₀ (50% growth inhibition) wasdetermined. Cells infected with no virus or N2 virus act as controlsamples for these experiments. The N2 viruses contain no VZV TK geneticmaterial.

[0234] Table 1 demonstrates that the HepG2 hepatoma cells infected witheither pCR74- or pCR78-containing viruses have approximately 700 fold or33 fold greater VZV TK activity, respectively, compared to controlcells. The HuH7 hepatoma cells infected with either pCR74- orpCR78-containing viruses have approximately 218-fold and 1 5-foldgreater VZV TK activity, respectively, compared to control cells.

[0235] 9-β-D-arabinofuranosyl-6-methoxy-9H-purine (designated as araM)can be selectively monophosphorylated by VZV TK with subsequentanabolism to cytotoxic ara-ATP. FIG. 9 demonstrates that HepG2 cellswhich were infected with either pCR74- or pCR78-containing viruses andincubated in the presence of (³H)-labelled9-β-D-arabinofuranosyl-6-methoxy-9H-purine had significant amounts ofcytotoxic (³H)-ara-ATP formation.

[0236] HepG2 and HuH7 cells infected with the replication-defective,full-length, artificial retroviruses containing the ALB/VZV TK chimaera(pCR74) or AFP/VZV TK (pCR78) chimaera were incubated in the presence ofvarying amounts of 9-β-D-arabinofuranosyl-6-methoxy-9H-purine for 5 daysand growth inhibition was determined as measured by cell number and DNAcontent.

[0237] Table 2 demonstrates that the IC₅₀ (50% growth inhibition) of9-β-D-arabinofuranosyl-6-methoxy-9H-purine (araM) is greater than 2,000uM in control and N2 infected HepG2 cells. The IC₅₀ of araM isapproximately 1621 uM in control HuH7 cells. In HepG2 cells infectedwith the replication-defective, full-length, artificial retrovirusescontaining the ALB/VZV TK chimaera (pCR74) or AFP/VZV TK (pCR78)chimaera, the IC₅₀ values were 6.5 uM and 78 uM, respectively. Singlecell cloning of HepG2 cells containing the AFP/VZV TK (pCR78) chimaeraindicated that the IC₅₀ levels of9-β-D-arabinofuranosyl-6-methoxy-9H-purine can be further decreased toapproximately 40 uM. In HuH7 cells infected with thereplication-defective, full-length, artificial retroviruses containingthe ALB/VZV TK chimaera (pCR74) or AFP/VZV TK (pCR78) chimaera, the IC₅₀values were 11 uM and 76 uM, respectively.

EXAMPLE 7 Selectivity of Expression of VZV TK

[0238] Four human, nonhepatoma cell lines were infected withreplication-defective, full-length, artifical retroviruses containingthe ALB/VZV TK chimaera (pCR74) or AFP/VZV TK chimaera (pCR78). Thesecell lines were WiDR (ATCC CCL218), MCF7 (ATCC HTB22), Detroit 555 (ATCCCCL110), and SW480 (ATCC CCL228). Subsequent to infection and selectionon Geneticin (antibiotic G418 sulphate; registered trademark of GIBCO),these cells were assayed for VZV TK activity and growth inhibition inthe presence of 9-β-D-arabinofuranosyl-6-methoxy-9H-purine, as describedabove. There was no increase in VZV TK activity or drug sensitivity to9-β-D-arabino-furanosyl-6-methoxy-9H-purine in these four nonhepatomacell lines infected with replication-defective, full-length, artificialretroviruses containing the ALB/VZV TK chimaera (pCR74) or AFP/VZV TKchimaera (pCR78) compared to parental cell lines which were notinfected. This demonstrates the selectivity of expression for VZV TK inhepatoma versus nonhepatoma cells.

EXAMPLE 8 Construction of Transcriptional Regulatory Sequence ofCarcinoembryonic Antigen/Cytosine Deaminase Molecular Chimaera

[0239] A) Cloning and Isolation of the Transcriptional RegulatorySequence of the Carcinoembryonic Antigen Gene

[0240] CEA genomic clones were identified and isolated from the humanchromosome 19 genomic library LL19NL01, ATCC #57766, by standardtechniques (Richards, C. A. et al. Cancer Research 50:1521-1527, 1990which is herein incorporated by reference in its entirety). The CEAclones were identified by plaque hybridization to ³²P end-labelledoligonucleotides CR15 and CR16. CR15,5′-CCCTGTGATCTCCAGGACAGCTCAGTCTC-3′(SEQ ID NO:7), and CR16,5′-GTTTCCTGAGTGATGTCTGTGTGCAATG-3′(SEQ ID NO:8), hybridize to a 5′non-transcribed region of CEA that has little homology to other membersof the CEA gene family. Phage DNA was isolated from three clones thathybridized to both oligonucleotide probes. Polymerase chain reaction,restriction mapping, and DNA sequence analysis confirmed that the threeclones contained CEA genomic sequences. The three clones are designatedlambdaCEA1, lambdaCEA5, and lambdaCEA7 and have inserts of approximately13.5, 16.2, and 16.7 kb respectively. A partial restriction map of thethree overlapping clones is shown in FIG. 10.

[0241] Clone lambdaCEA1 was initally chosen for extensive analysis.Fragments isolated from lambdaCEA1 were subcloned using standardtechniques into the plasmid pBS +(Stratagene Cloning Systems, La Jolla,Calif.) to facilitate sequencing, site-directed mutagenesis, andconstruction of chimeric genes. The inserts of some clones arerepresented in FIG. 11. The complete DNA sequence of a 11,288 bp HindIII/Sau3A restriction fragment from lambdaCEA1 (FIG. 12A, SEQ ID NO:4)was determined by the dideoxy sequencing method using the dsDNA CycleSequencing System from Life Technologies, Inc. and multipleoligonucleotide primers.

[0242] This sequence extends from −10.7 kb to +0.6 kb relative to thestart site of CEA mRNA. The sequence of 3774 base pair Hind IIIrestriction fragment from lambdaCEA1 was also determined (FIG. 12B SEQID NO:5). This sequence extends from −14.5 kb to −10.7 kb relative tothe start site of CEA mRNA. This Hind III fragment is present in plasmidpCR145.

[0243] To determine important transcriptional regulatory sequencesvarious fragments of CEA genomic DNA are linked to a reporter gene suchas luciferase or chloramphenicol acetyltransferase. Various fragments ofCEA genomic DNA are tested to determine the optimized, cell-typespecific TRS that results in high level reporter gene expression inCEA-positive cells but not in CEA-negative cells. The various reporterconstructs, along with appropriate controls, are transfected into tissueculture cell lines that express high, low, or no CEA. The reporter geneanalysis identifies both positive and negative transcriptionalregulatory sequences. The optimized CEA-specific TRS is identifiedthrough the reporter gene analysis and is used to specifically directthe expression of any desired linked coding sequence, such as cytosinedeaminase or VZV TK, in cancerous cells that express CEA. The optimizedCEA-specfic TRS, as used herein, refers to any DNA construct thatdirects suitably high levels of expression in CEA positive cells and lowor no expression in CEA-negative cells. The optimized CEA-specific TRSconsists of one or several different fragments of CEA genomic sequenceor multimers of selected sequences that are linked together by standardrecombinant DNA techniques. It will be appreciated by those skilled inthe art that the optimized CEA-specific TRS may also include somesequences that are not derived from the CEA genomic sequences shown inFIGS. 12A or 12B. These other sequences may include sequences fromadjoining regions of the CEA locus, such as sequences from the introns,or sequences further upstream or downstream from the sequenced DNA shownin FIGS. 12A or 12B, or they could include transcriptional controlelements from other genes that when linked to selected CEA sequencesresult in the desired CEA-specific regulation.

[0244] The CEA sequence of FIGS. 12A and 12B were computer analyzed forcharacterized consensus sequences which have been associated with generegulation. Currently not enough is known about transcriptionalregulatory sequences to accurately predict by sequence alone whether asequence will be functional. However, computer searches forcharacterized consensus sequences can help identify transcriptionalregulatory sequences in uncharacterized sequences since many enhancersand promoters consist of unique combinations and spatial alignments ofseveral characterized consensus sequences as well as other sequences.Since not all transcriptional regulatory sequences have been identifiedand not all sequences that are identical to characterized consensussequences are functional, such a computer analysis can only suggestpossible regions of DNA that may be functionally important for generegulation.

[0245] Some examples of the consensus sequences that are present in theCEA sequence (FIGS. 12A and 12B) are shown in FIG. 12C. Four copies of alysozymal silencer consensus sequences have been found in the CEAsequence. Inclusion of one or more copies of this consensus sequence inthe molecular chimera can help optimize CEA-specific expression. Acluster of topoisomerase 11 cleavage consensus identified approximately4-5 kb upstream of the CEA transcriptional start suggest that thisregion of CEA sequence may contain important transcriptional regulatorysignals that may help optimize CEA-specific expression.

[0246] The first fragment of CEA genomic sequence analyzed fortranscriptional activity extends from −299 to +69, but it is appreciatedby those skilled in the art that other fragments are tested in order toisolate a TRS that directs strong expression in CEA-positive cells butlittle expression in CEA-negative cells. As diagrammed in FIG. 13 the943 bp SmaI-Hind III fragment of plasmid 39-5-5 was subcloned into theSmaI-HindIII sites of vector pBS+(Statagene Cloning Systems) creatingplasmid 96-11. Single-stranded DNA was rescued from cultures of XL1-blue96-11 using an M13 helper virus by standard techniques. OligonucleotideCR70, 5′-CCTGGAACTCAAGCTTGAATTCTCCACAGAGGAGG-3′(SEQ ID NO:9), was usedas a primer for oligonucleotide-directed mutagenesis to introduceHindIII and EcoRI restriction sites at +65. Clone 109-3 was isolatedfrom the mutagenesis reaction and was verified by restriction and DNAsequence analysis to contain the desired changes in the DNA sequence.CEA genomic sequences -299 to +69, original numbering FIG. 12, wereisolated from 109-3 as a 381 bp EcoR I/Hind III fragment. PlasmidpRc/CMV (Invitrogen Corporation, San Diego, Calif.) was restricted withAat II and Hind III and the 4.5 kb fragment was isolated from lowmelting point agarose by standard techniques. The 4.5 kb fragment ofpRc/CMV was ligated to the 381 bp fragment of 109-3 using T4 DNA ligase.During this ligation the compatible Hind III ends of the two differentrestriction fragments were ligated. Subsequently the ligation reactionwas supplemented with the four deoxynucleotides, dATP, dCTP, dGTP, anddTTP, and T4 DNA polymerase in order to blunt the non-compatible Aat IIand EcoR I ends. After incubating, phenol extracting, and ethanolprecipitating the reaction, the DNAs were again incubated with T4 DNAligase. The resulting plasmid, pCR92, allows the insertion of anydesired coding sequence into the unique Hind III site downstream of theCEA TRS, upstream from a polyadenylation site and linked to a dominantselectable marker. The coding sequence for cytosine deaminase or otherdesirable effector or reporter gene, when inserted in the correctorientation into the HindIII site, are transcriptionally regulated bythe CEA sequences and are preferably expressed in cells that express CEAbut not in cells that do not express CEA.

[0247] In order to determine the optimized CEA TRS other reporter geneconstructs containing various fragments of CEA genomic sequences aremade by standard techniques from DNA isolated from any of the CEAgenomic clones (FIGS. 10, 11, 13, and 14). DNA fragments extending fromthe HindIII site introduced at position +65 (original numbering FIG.12A) and numerous different upstream sites are isolated and cloned intothe unique HindIII site in plasmid pSVOALdelta5′ (De Wet, J. R., et al.Molecular and Cellular Biology 7:725-737, 1987 which is hereinincorporated by reference in its entirety) or any similar reporter geneplasmid to construct luciferase reporter gene constructs, FIGS. 13 and14. These and similar constructs are used in transient expression assaysperformed in several CEA-positive and CEA-negative cell lines todetermine a strong, CEA-positive cell-type specific TRS. FIGS. 14B, 14C,and 14D show the results obtained from several CEA/luciferase reporterconstructs. The optimized TRS is used to regulate the expression ofcytosine deaminase or other desirable gene in a cell-type specificpattern in order to be able to specifically kill cancer cells. Thedesirable expression cassette is added to a retroviral shuttle vector toaid in delivery of the expression cassette to cancerous tissue.

[0248] Strains containing plasmids 39-5-5 and 39-5-2 were deposited atthe ATCC under the Budapest Treaty with Accession No. 68904 and 68905,respectively. A strain containing plasmid pCR92 was deposited with theATCC under the Budapest Treaty with Accession No. 68914. A straincontaining plasmid pCR145 was deposited at the ATCC under the BudapestTreaty with Accession No. 69460.

[0249] B) Cloning and Isolation of the E. coli Gene Encoding CytosineDeaminase

[0250] A positive genetic selection was designed for the cloning of thecodA gene from E. coli. The selection took advantage of the fact that E.coli is only able to metabolize cytosine via cytosine deaminase. Basedon this, an E. coli strain was constructed that could only utilizecytosine as a pyrimidine source when cytosine deaminase was provided intrans. This strain, BA101, contains a deletion of the codAB operon and amutation in the pyrF gene. The strain was created by transducing a pyrFmutation (obtained from the E. coli strain X82 (E. coli Genetic StockCenter, New Haven, Conn.)) into the strain MBM7007 (W. Dallas, BurroughsWellcome Co., North Carolina) which carried a deletion of the chromosomefrom Iac to argF. The pyrF mutation confers a pyrimidine requirement onthe strain, BA101. In addition, the strain is unable to metabolizecytosine due to the codAB deletion. Thus, BA101 is able to grow onminimal medium supplemented with uracil but is unable to utilizecytosine as the sole pyrimidine source. This is illustrated in FIG. 15.

[0251] The construction of BA101 provided a means for positive selectionof DNA fragments encoding cytosine deaminase. The strain, BA101, wastransformed with plasmids carrying inserts from the E. coli chromosomeand the transformants were selected for growth on minimal mediumsupplemented with cytosine. Using this approach, the transformants werescreened for the ability to metabolize cytosine indicating the presenceof a DNA fragment encoding cytosine deaminase. Several sources of DNAcould be used for the cloning of the codA gene: 1) a library of the E.coli chromosome could be purchased commercially (for example fromClontech, Palo Alto, Calif. or Stratagene, La Jolla, Calif.) andscreened; 2) chromosomal DNA could be isolated from E. coli, digestedwith various restriction enzymes and ligated and plasmid DNA withcompatible ends before screening; and/or 3) bacteriophage lambda clonescontaining mapped E. coli chromosomal DNA inserts could be screened.

[0252] Bacteriophage lambda clones (Y. Kohara, National Institute ofGenetics, Japan) containing DNA inserts spanning the 6-8 minute regionof the E. coli chromosome were screened for the ability to providetransient complementation of the codA defect. Two clones, 137 and 138were identified in this manner. Large-scale preparations of DNA fromthese clones were isolated from 500 ml cultures. Restriction enzymeswere used to generate DNA fragments ranging in size from 10-12kilobases. The enzymes used were EcoRI, EcoRI and BamHI, and EcoRI andHindIII. DNA fragments of the desired size were isolated frompreparative agarose gels by electroelution. The isolated fragments wereligated to pBR322 (Gibco BRL, Gaithersburg) with compatible ends. Theresulting ligation reactions were used to transform the E. coli strain,DH5a (Gibco BRL, Gaithersburg, Md.). This step was used to amplify therecombinant plasmids resulting from the ligation reactions. The plasmidDNA preparations isolated from the ampicillin-resistant DH5αtransformants were digested with the appropriate restriction enzymes toverify the presence of insert DNA. The isolated plasmid DNA was used totransform BA101. The transformed cells were selected for resistance toampicillin and for the ability to metabolize cytosine. Two clones wereisolated pEA001 (FIG. 16) and pEA002 (FIG. 17). The plasmid pEA001contains an approximately 10.8 kb EcoRI-BamHI insert while pEA002contains an approximately 11.5 kb EcoRI-HindIII insert. The isolatedplasmids were used to transform BA101 to ensure that the ability tometabolize cytosine was the result of the plasmid and not due to aspontaneous chromosomal mutation.

[0253] A physical map of the pEA001 DNA insert was generated usingrestriction enzymes. Deletion derivatives of pEA001 were constructedbased on this restriction map (FIG. 18). The resulting plasmids werescreened for the ability to allow BA101 to metabolize cytosine. Usingthis approach, the codA gene was localized to a 4.8 kb EcorI-BgAIIfragment (FIG. 19). The presence of codA within these inserts wasverified by enzymatic assays for cytosine deaminase activity (FIG. 18).In addition, cell extracts prepared for enzymatic assay were alsoexamined by polyacrylamide gel electrophoresis. Cell extracts that werepositive for enzymatic activity also had a protein band migrating withan apparent molecular weight of 52,000 (FIG. 20).

[0254] The DNA sequence of both strands was determined for a 1634basepair fragment (FIG. 21). The sequence determination began at thePstI site and extended to PvuII site thus including the codA codingdomain (SEQ ID NO:6). An open reading frame of 1283 nucleotides wasidentified. The thirty amino terminal amino acids were confirmed byprotein sequencing. Additional internal amino acid sequences weregenerated from CNBr-digestion of gel-purified cytosine deaminase. Theamino acids verified by protein sequencing are underlined in FIG. 21.

[0255] A 200 basepair PstI fragment was isolated that spanned thetranslational start codon of codA. This fragment was cloned into pBS⁺.Single-stranded DNA was isolated from 30 ml culture and mutanized usingthe custom oligonuclotide BA22 (Sequence: 5′-GACGCATGTGGAAGCTTACAATGTCGAATAACGC-3′(SEQ ID NO:10)) purchased from Synthecell Inc., Rockville,Md., and the oligonucleotide-directed mutagenesis kit (Amersham,Arlington Heights, Ill.). The underlined bases in the sequence of theBA22 oligonucleotide represent base changes introduced by themutagenesis. These changes result in the introduction of an HindIIIrestriction enzyme site for joining of cytosine deaminase with CEA TRSand in a translational start codon of ATG rather than GTG. The resulting90 basepair HindIII-PstI fragment is isolated and ligated with theremainder of the cytosine deaminase gene. The chimeric CEA TRS/cytosinedeaminase gene is created by ligating the HindIII-PvuII cytosinedeaminase-containing DNA fragment with the CEA TRS sequences.

[0256] The strain BA101 and the plasmids, pEA001 and pEA003, weredeposited with ATCC under the Budapest Treaty with Accession Nos. 55299,68916, and 68915 respectively.

[0257] C) Construction of Transcriptional Regulatory Sequence ofCarcinoembryonic Antigen/Cytosine Deaminase Molecular Chimera

[0258] A 1508 bp HindIII/PvuII fragment containing the coding sequencefor cytosine deaminase is isolated from the plasmid containing the fulllength cytosine deaminase gene of Example 69B that has been altered tocontain a HindIII restriction site just 5 ′ of the initation codon.Plasmid pCR92 contains CEA sequences −299 to +69 immediately 5′ to aunique HindIII restriction site and a polyadenylation signal 3′ to aunique ApaI restriction site (Example 8A, FIG. 13). pCR92 is lineraizedwith Apa I, the ends are blunted using dNTPs and T4 DNA polymerase, andsubsequently digested with HindIII. The pCR92 HindIII/ApaI fragment isligated to the 1508 bp HindIII/PvuII fragment containing cytosinedeaminase. Plasmid pCEA-1/codA, containing cytosine deaminase insertedin the appropriate orientation relative to the CEA TRS andpolyadenylation signal is identifed by restriction enzyme and DNAsequence analysis.

[0259] The optimized CEA-specific TRS, the coding sequence for cytosinedeaminase with an ATG translation start, and a suitable polyadenylationsignal are joined together using standard molecular biology techniques.The resulting plasmid, containing cytosine deaminase inserted in theappropriate orientation relative to the optimized CEA specific TRS and apolyadenylation signal is identified by restriction enzyme and DNAsequence analysis.

EXAMPLE 9 Construction of a Retroviral Shuttle Vector ConstructContaining the Molecular Chimera of Example 8

[0260] The retroviral shuttle vector pL-CEA-1/codA is constructed byligating a suitable restriction fragment containing the optimized CEATRS/codA molecular chimera including the polyadenylation signal into anappropriate retroviral shuttle vector, such as N2 (XM5) linearized atthe Xho I site, using standard molecular biology techniques similar tothose detailed in Examples 3 and 4. The retroviral shuttle vectorpL-CEA-1/codA is characterized by restriction endonuclease mapping andpartial DNA sequencing.

EXAMPLE 10 Virus Production of Retroviral Constructs of Example 9

[0261] The retroviral shuttle construct described in Example 9 is placedinto an appropriate packaging cell line, such as PA317, byelectroporation or infection as described in Example 5. Drug resistantcolonies, such as those resistant to G418 when using shuttle vectorscontaining the NEO gene, are single cell cloned by the limiting dilutionmethod, analyzed by Southern blots, and titred in NIH 3T3 cells toidentify the highest producer of full-length virus.

EXAMPLE 11 Demonstration of Neighboring Cell Killing Effect

[0262] The following data illustrates and supports this importantcomponent of this invention. A human colorectal carcinoma cell line,WiDr, was genetically engineered to express cytosine deaminase (CD) bytransfecting into the cell line the cloned gene for cytosine deaminasein an appropriate expression vector system for mammalian cells. WiDrcells expressing cytosine deaminase (WiDR/CD) and control cells notexpressing cytosine deaminase (WiDr) were mixed together at differentratios, then plated at a total of 3,000 cells per well in a 96 wellmicrotiter dish. Growth kinetics of these cells over an 8 day periodindicated that the different mixtures of cells all grew at approximatelyequal rates (FIG. 22). This data confirmed that the growth rates of thedifferent mixtures of cells were indistinguishable. Using the samemixtures of WiDr and WiDr/CD cells and again plating 3,000 total cellsper well in a 96 well microtiter dish, log dose response curves weregenerated for the inhibition of cell growth by 5-FC. FIG. 23 indicatesthat 5-FC was very nontoxic to control WiDr cells (IC₅₀ between 10,000and 30,000 uM) but very toxic to WiDR/CD cells (IC₅₀ between 10 uM and25 uM). Importantly, all the different mixtures of cells showed toxicitypatterns similar to the WiDr/CD cells. These data indicate that WiDr/CDcells generated sufficient toxic metabolites of 5-FC to inhibit the cellgrowth of neighboring cells.

[0263] The same mixtures of WiDr and WiDr/CD cells were injectedsubcutaneously into 10 individual nude mice for 10 individual injectionsof 10 million cells for each mixture (Table 3). By day 6, all mice hadtumors of approximately the same size. On day 6, 5 mice of each groupreceived a daily injection of 5-FC (ip at 500mg/kg body weight) forapproximately 20 consecutive days, then injections of 5-FC (ip at 500mg/kg body weight) three times a week for 19 days. By 22 days post tumorcell injection, all nontreated animals had to be killed due to the largesize of the tumors. Likewise, all animals with WiDr derived tumors whichalso received 5-FC treatment had to be sacrificed on day 22 due to thesize of the tumors. This indicates that 5-FC has no effect on WiDrtumors that do not express cytosine deaminase. However, in all tumorgroups composed of mixtures of cells which contained WiDr/CD cells,there were significant antitumor effects due to 5-FC treatment. In allgroups there were between 3 out of 5, to 4 out of 5 tumor cures (asdefined as being tumor free by day 130). This indicates that theindicated percentage of WiDr/CD cells in a mixed tumor generatedsufficient toxic metabolites of 5-FC to kill all WiDr and WiDr/CD cellsin the tumor.

[0264] The following example illustrates pharmaceutical formulations(compositions) which are in accordance with the present invention.

EXAMPLE 12

[0265] Injectable Formulation Infective virion 2 × 10⁶ Colony FormingUnits (CFU) Physiologic aqueous solution 1 mL

[0266] The infective virion as described herein is asceptically admixedwith the a physiologic aqueous solution, which may or may not containstabilizers, in a suitable sterile glass vial and sealed with a sterileclosure and overseal. Injectable Formulation Packaging cell line 2 × 10⁶Cells Physiologic aqueous solution 1 mL

[0267] Cells of the packaging cell line as described herein areasceptically admixed with the physiologic aqueous solution, which may ormay not contain stabilizers, in a suitable sterile glass vial and sealedwith a sterile closure and overseal. TABLE 1 VZV TK activity in HepG2and HuH7 cells infected with replication-defective, full-length,artificial retroviruses containing ALB/VZV TK chimaera (pCR74) orAFP/VZV TK chimaera (pCR78). VZV TK activity was quantitated as amountof araM phosphorylated per mg cellular protein per 30 minutes. VZV TKEnzymatic Activity pMoles araM phosphate/mg protein/30 mins Virus HepG2HuH7 None   9  13 N2   4 N.D. pCR74 4521 2831 pCR78  198  200

[0268] TABLE 2 Growth inhibition in HepG2 and HuH7 cells infected withreplication-defective, full-length, artificial retroviruses containingALB/VZV TK chimaera (pCR74) or AFP/VZV TK chimaera (pCR78). IC50 for9-B-D-arabinofuranosyl-6-methoxy- 9H-purine Virus HepG2 HuH7 None >2000uM 1621 uM N2 >2000 uM N.D. pCR74 5 uM 11 uM pCR78 175 uM 76 uM

[0269] TABLE 3 Number of Cures Treatment Cells Injected None 5-FC WiDR0/5 (22)¹ 0/5  (22) WiDr/CD 0/5 (22) 3/5 (130) WiDr:WiDr/CD (2:1) 0/5(22) 4/5 (130) WiDr:WiDr/CD (1:1) 0/5 (22) 4/5 (130) WiDr:WiDr/CD (1:2)0/5 (22) 4/5 (130)

[0270]

1 36 1 1980 DNA Varicella zoster 1 cctgtaacag gttcagaccc cgttgagatacaaacacaag gaggggggtc accattattt 60 catcagatcc cgtgggtgtg gtttcctttattaaagccat ggtatccctc agctggcgca 120 taccctcgca aaactggtga tacttagtaggggtatgtat attagcgcta aaacggcaag 180 attttaattc cactataaaa caaacggtctttccggcacc actggattcc gtttgtataa 240 tacaaacaca atcggggcgt cggcgtcccaaatttacttc aaacgacatt gatatgcgta 300 cagccctttg aacatccacg tgggataacggcgacaggag ttttgccagc ctcgggttga 360 acgcgtccgc gaaacctcga cgtacgttatcaatatcctt tttgagtaca tcgtaaaaac 420 gagtgtggca acgttgtccc aaacgaaaacacttggcccg aattcgacta gcggacatat 480 ttgaagttcc gtcccagaag ataacctaagacgcgtttgt ctacaataaa catgtcaacg 540 gataaaaccg atgtaaaaat gggcgttttgcgtatttatt tggacggggc gtatggaatt 600 ggaaaaacga ccgccgccga agaatttttacaccactttg caataacacc aaaccggatc 660 ttactcattg gggagcccct gtcgtattggcgtaaccttg caggggagga cgccatttgc 720 ggaatttacg gaacacaaac tcgccgtcttaatggagacg tttcgcctga agacgcacaa 780 cgcctcacgg ctcattttca gagcctgttctgttctccgc atgcaattat gcatgcgaaa 840 atctcggcat tgatggacac aagtacatcggatctcgtac aagtaaataa ggagccgtat 900 aaaattatgt tatccgaccg acacccaatcgcctcaacta tatgttttcc cttgtccaga 960 tacttagtgg gagatatgtc cccagcggcgcttcctgggt tattgtttac gcttcccgct 1020 gaaccccccg ggaccaactt ggtagtttgtaccgtttcac tccccagtca tttatccaga 1080 gtaagcaaac gggccagacc gggagaaacggttaatctgc cgtttgttat ggttctgaga 1140 aatgtatata taatgcttat taatacaattatatttctta aaactaacaa ctggcacgcg 1200 ggctggaaca cactgtcatt ttgtaatgatgtatttaaac agaaattaca aaaatccgag 1260 tgtataaaac tacgcgaagt acctgggattgaagacacgt tattcgccgt gcttaaactt 1320 ccggagcttt gcggagagtt tggaaatattctgccgttat gggcatgggg aatggagacc 1380 ctttcaaact gcttacgaag catgtctccgttcgtattat cgttagaaca gacaccccag 1440 catgcggcac aagaactaaa aactctgctaccccagatga ccccggcaaa catgtcctcc 1500 ggtgcatgga atatattgaa agagcttgttaatgccgttc aggacaacac ttcctaaata 1560 tacctagtat ttacgtatgt accagtaaaaagatgataca cattgtcata ctcgcgtgta 1620 cgtgtttttc ttttttatat atgcgtcatttattaccaca tcctttaatc ccgcctttat 1680 ctccctaaaa cggagtggta atattaaaagccgccaagcc tgttggtggg tgaggagggg 1740 taaaggcacg ctgtgtgcat aacgttgcggtgatattgta gcgcaagtaa cagcgactat 1800 gtttgcgcta gttttagcgg tggtaattcttcctctgtgg accacggcta ataaatctta 1860 cgtaacacca acccctgcga ctcgctctatcggacatatg tctgctcttc tacgagaata 1920 ttccgaccgt aatatgtctc tgaaattagaagccttttat cctactggtt tcgatgaaga 1980 2 276 DNA Artificial sequencemisc_feature Description of artificial sequence chimera of murine ALBand var icella zoster virus thymidine kinase. 2 atggtatgat tttgtaatggggtaggaacc aatgaaatgc gaggtaagta tggttaatga 60 tctacagtta ttggttaaagaagtatatta gagcgagtct ttctgcacac agatcacctt 120 tcctatcaac cccgggatcctacaataaac atgtcaacgg ataaaaccga tgtaaaaatg 180 ggcgttttgc gtatttatttggacggggcg tatggaattg gaaaaacgac cgccgccgaa 240 gaatttttac accactttgcaataacacca aaccgg 276 3 228 DNA Artificial sequence misc_featureDescription of artificial sequence chimera of human AFP and vari cellazoster virus thymidine kinase 3 gcattgcctg aaaagagtat aaaagaatttcagcatgatt ttccatattg tgcttccacc 60 actgccaata acaccggatc gcaagctgatcctacaataa acatgtcaac ggataaaacc 120 gatgtaaaaa tgggcgtttt gcgtatttatttggacgggg cgtatggaat tggaaaaacg 180 accgccgccg aagaattttt acaccactttgcaataacac caaaccgg 228 4 11288 DNA Homo sapiens 4 aagcttaaaa cccaatggattgacaacatc aagagttgga acaagtggac atggagatgt 60 tacttgtgga aatttagatgtgttcagcta tcgggcagga gaatctgtgt caaattccag 120 catggttcag aagaatcaaaaagtgtcaca gtccaaatgt cgaacagtgc aggggataaa 180 actgtggtgc attcaaactgagggatattt tggaacatga gaaaggaagg gattgctgct 240 gcacagaaca tggatgatctcacacataga gttgaaagaa aggagtcaat cgcagaatag 300 aaaatgatca ctaattccacctctataaag tttccaagag gaaaacccaa ttctgctgct 360 agagatcaga atggaggtgacctgtgcctt gcaatggctg tgagggtcac gggagtgtca 420 cttagtgcag gcaatgtgccgtatcttaat ctgggcaggg ctttcatgag cacataggaa 480 tgcagacatt actgctgtgttcattttact tcaccggaaa agaagaataa aatcagccgg 540 gcgcggtggc tcacgcctgtaatcccagca ctttagaagg ctgaggtggg cagattactt 600 gaggtcagga gttcaagaccaccctggcca atatggtgaa accccggctc tactaaaaat 660 acaaaaatta gctgggcatggtggtgcgcg cctgtaatcc cagctactcg ggaggctgag 720 gctggacaat tgcttggacccaggaagcag aggttgcagt gagccaagat tgtgccactg 780 cactccagct tgggcaacagagccagactc tgtaaaaaaa aaaaaaaaaa aaaaaaaaag 840 aaagaaagaa aaagaaaagaaagtataaaa tctctttggg ttaacaaaaa aagatccaca 900 aaacaaacac cagctcttatcaaacttaca caactctgcc agagaacagg aaacacaaat 960 actcattaac tcacttttgtggcaataaaa ccttcatgtc aaaaggagac caggacacaa 1020 tgaggaagta aaactgcaggccctacttgg gtgcagagag ggaaaatcca caaataaaac 1080 attaccagaa ggagctaagatttactgcat tgagttcatt ccccaggtat gcaaggtgat 1140 tttaacacct gaaaatcaatcattgccttt actacataga cagattagct agaaaaaaat 1200 tacaactagc agaacagaagcaatttggcc ttcctaaaat tccacatcat atcatcatga 1260 tggagacagt gcagacgccaatgacaataa aaagagggac ctccgtcacc cggtaaacat 1320 gtccacacag ctccagcaagcacccgtctt cccagtgaat cactgtaacc tcccctttaa 1380 tcagccccag gcaaggctgcctgcgatggc cacacaggct ccaacccgtg ggcctcaacc 1440 tcccgcagag gctctcctttggccacccca tggggagagc atgaggacag ggcagagccc 1500 tctgatgccc acacatggcaggagctgacg ccagagccat gggggctgga gagcagagct 1560 gctggggtca gagcttcctgaggacaccca ggcctaaggg aaggcagctc cctggatggg 1620 ggcaaccagg ctccgggctccaacctcaga gcccgcatgg gaggagccag cactctaggc 1680 ctttcctagg gtgactctgaggggaccctg acacgacagg atcgctgaat gcacccgaga 1740 tgaaggggcc accacgggaccctgctctcg tggcagatca ggagagagtg ggacaccatg 1800 ccaggccccc atggcatggctgcgactgac ccaggccact cccctgcatg catcagcctc 1860 ggtaagtcac atgaccaagcccaggaccaa tgtggaagga aggaaacagc atccccttta 1920 gtgatggaac ccaaggtcagtgcaaagaga ggccatgagc agttaggaag ggtggtccaa 1980 cctacagcac aaaccatcatctatcataag tagaagccct gctccatgac ccctgcattt 2040 aaataaacgt ttgttaaatgagtcaaattc cctcaccatg agagctcacc tgtgtgtagg 2100 cccatcacac acacaaacacacacacacac acacacacac acacacacac acacagggaa 2160 agtgcaggat cctggacagcaccaggcagg cttcacaggc agagcaaaca gcgtgaatga 2220 cccatgcagt gccctgggccccatcagctc agagaccctg tgagggctga gatggggcta 2280 ggcaggggag agacttagagagggtggggc ctccagggag ggggctgcag ggagctgggt 2340 actgccctcc agggagggggctgcagggag ctgggtactg ccctccaggg agggggctgc 2400 agggagctgg gtactgccctccagggaggg ggctgcaggg agctgggtac tgccctccag 2460 ggagggggct gcagggagctgggtactgcc ctccagggag gcaggagcac tgttcccaac 2520 agagagcaca tcttcctgcagcagctgcac agacacagga gcccccatga ctgccctggg 2580 ccagggtgtg gattccaaatttcgtgcccc attgggtggg acggaggttg accgtgacat 2640 ccaaggggca tctgtgattccaaacttaaa ctactgtgcc tacaaaatag gaaataaccc 2700 tactttttct actatctcaaattccctaag cacaagctag caccctttaa atcaggaagt 2760 tcagtcactc ctggggtcctcccatgcccc cagtctgact tgcaggtgca cagggtggct 2820 gacatctgtc cttgctcctcctcttggctc aactgccgcc cctcctgggg gtgactgatg 2880 gtcaggacaa gggatcctagagctggcccc atgattgaca ggaaggcagg acttggcctc 2940 cattctgaag actaggggtgtcaagagagc tgggcatccc acagagctgc acaagatgac 3000 gcggacagag ggtgacacagggctcagggc ttcagacggg tcgggaggct cagctgagag 3060 ttcagggaca gacctgaggagcctcagtgg gaaaagaagc actgaagtgg gaagttctgg 3120 aatgttctgg acaagcctgagtgctctaag gaaatgctcc caccccgatg tagcctgcag 3180 cactggacgg tctgtgtacctccccgctgc ccatcctctc acagcccccg cctctaggga 3240 cacaactcct gccctaacatgcatctttcc tgtctcattc cacacaaaag ggcctctggg 3300 gtccctgttc tgcattgcaaggagtggagg tcacgttccc acagaccacc cagcaacagg 3360 gtcctatgga ggtgcggtcaggaggatcac acgtcccccc atgcccaggg gactgactct 3420 gggggtgatg gattggcctggaggccactg gtcccctctg tccctgaggg gaatctgcac 3480 cctggaggct gccacatccctcctgattct ttcagctgag ggcccttctt gaaatcccag 3540 ggaggactca acccccactgggaaaggccc agtgtggacg gttccacagc agcccagcta 3600 aggcccttgg acacagatcctgagtgagag aacctttagg gacacaggtg cacggccatg 3660 tccccagtgc ccacacagagcaggggcatc tggaccctga gtgtgtagct cccgcgactg 3720 aacccagccc ttccccaatgacgtgacccc tggggtggct ccaggtctcc agtccatgcc 3780 accaaaatct ccagattgagggtcctccct tgagtccctg atgcctgtcc aggagctgcc 3840 ccctgagcaa atctagagtgcagagggctg ggattgtggc agtaaaagca gccacatttg 3900 tctcaggaag gaaagggaggacatgagctc caggaagggc gatggcgtcc tctagtgggc 3960 gcctcctgtt aatgagcaaaaaggggccag gagagttgag agatcagggc tggccttgga 4020 ctaaggctca gatggagaggactgaggtgc aaagaggggg ctgaagtagg ggagtggtcg 4080 ggagagatgg gaggagcaggtaaggggaag ccccagggag gccgggggag ggtacagcag 4140 agctctccac tcctcagcattgacatttgg ggtggtcgtg ctagtggggt tctgtaagtt 4200 gtagggtgtt cagcaccatctggggactct acccactaaa tgccagcagg actccctccc 4260 caagctctaa caaccaacaatgtctccaga ctttccaaat gtcccctgga gagcaaaatt 4320 gcttctggca gaatcactgatctacgtcag tctctaaaag tgactcatca gcgaaatcct 4380 tcacctcttg ggagaagaatcacaagtgtg agaggggtag aaactgcaga cttcaaaatc 4440 tttccaaaag agttttacttaatcagcagt ttgatgtccc aggagaagat acatttagag 4500 tgtttagagt tgatgccacatggctgcctg tacctcacag caggagcaga gtgggttttc 4560 caagggcctg taaccacaactggaatgaca ctcactgggt tacattacaa agtggaatgt 4620 ggggaattct gtagactttgggaagggaaa tgtatgacgt gagcccacag cctaaggcag 4680 tggacagtcc actttgaggctctcaccatc taggagacat ctcagccatg aacatagcca 4740 catctgtcat tagaaaacatgttttattaa gaggaaaaat ctaggctaga agtgctttat 4800 gctctttttt ctctttatgttcaaattcat atacttttag atcattcctt aaagaagaat 4860 ctatccccct aagtaaatgttatcactgac tggatagtgt tggtgtctca ctcccaaccc 4920 ctgtgtggtg acagtgccctgcttccccag ccctgggccc tctctgattc ctgagagctt 4980 tgggtgctcc ttcattaggaggaagagagg aagggtgttt ttaatattct caccattcac 5040 ccatccacct cttagacactgggaagaatc agttgcccac tcttggattt gatcctcgaa 5100 ttaatgacct ctatttctgtcccttgtcca tttcaacaat gtgacaggcc taagaggtgc 5160 cttctccatg tgatttttgaggagaaggtt ctcaagataa gttttctcac acctctttga 5220 attacctcca cctgtgtccccatcaccatt accagcagca tttggaccct ttttctgtta 5280 gtcagatgct ttccacctcttgagggtgta tactgtatgc tctctacaca ggaatatgca 5340 gaggaaatag aaaaagggaaatcgcattac tattcagaga gaagaagacc tttatgtgaa 5400 tgaatgagag tctaaaatcctaagagagcc catataaaat tattaccagt gctaaaacta 5460 caaaagttac actaacagtaaactagaata ataaaacatg catcacagtt gctggtaaag 5520 ctaaatcaga tatttttttcttagaaaaag cattccatgt gtgttgcagt gatgacagga 5580 gtgcccttca gtcaatatgctgcctgtaat ttttgttccc tggcagaatg tattgtcttt 5640 tctcccttta aatcttaaatgcaaaactaa aggcagctcc tgggccccct ccccaaagtc 5700 agctgcctgc aaccagccccacgaagagca gaggcctgag cttccctggt caaaataggg 5760 ggctagggag cttaaccttgctcgataaag ctgtgttccc agaatgtcgc tcctgttccc 5820 aggggcacca gcctggagggtggtgagcct cactggtggc ctgatgctta ccttgtgccc 5880 tcacaccagt ggtcactggaaccttgaaca cttggctgtc gcccggatct gcagatgtca 5940 agaacttctg gaagtcaaattactgcccac ttctccaggg cagatacctg tgaacatcca 6000 aaaccatgcc acagaaccctgcctggggtc tacaacacat atggactgtg agcaccaagt 6060 ccagccctga atctgtgaccacctgccaag atgcccctaa ctgggatcca ccaatcactg 6120 cacatggcag gcagcgaggcttggaggtgc ttcgccacaa ggcagcccca atttgctggg 6180 agtttcttgg cacctggtagtggtgaggag ccttgggacc ctcaggatta ctccccttaa 6240 gcatagtggg gacccttctgcatccccagc aggtgccccg ctcttcagag cctctctctc 6300 tgaggtttac ccagacccctgcaccaatga gaccatgctg aagcctcaga gagagagatg 6360 gagctttgac caggagccgctcttccttga gggccagggc agggaaagca ggaggcagca 6420 ccaggagtgg gaacaccagtgtctaagccc ctgatgagaa cagggtggtc tctcccatat 6480 gcccatacca ggcctgtgaacagaatcctc cttctgcagt gacaatgtct gagaggacga 6540 catgtttccc agcctaacgtgcagccatgc ccatctaccc actgcctact gcaggacagc 6600 accaacccag gagctgggaagctgggagaa gacatggaat acccatggct tctcaccttc 6660 ctccagtcca gtgggcaccatttatgccta ggacacccac ctgccggccc caggctctta 6720 agagttaggt cacctaggtgcctctgggag gccgaggcag gagaattgct tgaacccggg 6780 aggcagaggt tgcagtgagccgagatcaca ccactgcact ccagcctggg tgacagaatg 6840 agactctgtc tcaaaaaaaaagagaaagat agcatcagtg gctaccaagg gctaggggca 6900 ggggaaggtg gagagttaatgattaatagt atgaagtttc tatgtgagat gatgaaaatg 6960 ttctggaaaa aaaaatatagtggtgaggat gtagaatatt gtgaatataa ttaacggcat 7020 ttaattgtac acttaacatgattaatgtgg catattttat cttatgtatt tgactacatc 7080 caagaaacac tgggagagggaaagcccacc atgtaaaata cacccaccct aatcagatag 7140 tcctcattgt acccaggtacaggcccctca tgacctgcac aggaataact aaggatttaa 7200 ggacatgagg cttcccagccaactgcaggt gcacaacata aatgtatctg caaacagact 7260 gagagtaaag ctgggggcacaaacctcagc actgccagga cacacaccct tctcgtggat 7320 tctgacttta tctgacccggcccactgtcc agatcttgtt gtgggattgg gacaagggag 7380 gtcataaagc ctgtccccagggcactctgt gtgagcacac gagacctccc caccccccca 7440 ccgttaggtc tccacacatagatctgacca ttaggcattg tgaggaggac tctagcgcgg 7500 gctcagggat cacaccagagaatcaggtac agagaggaag acggggctcg aggagctgat 7560 ggatgacaca gagcagggttcctgcagtcc acaggtccag ctcaccctgg tgtaggtgcc 7620 ccatccccct gatccaggcatccctgacac agctccctcc cggagcctcc tcccaggtga 7680 cacatcaggg tccctcactcaagctgtcca gagagggcag caccttggac agcgcccacc 7740 ccacttcact cttcctccctcacagggctc agggctcagg gctcaagtct cagaacaaat 7800 ggcagaggcc agtgagcccagagatggtga cagggcaatg atccaggggc agctgcctga 7860 aacgggagca ggtgaagccacagatgggag aagatggttc aggaagaaaa atccaggaat 7920 gggcaggaga ggagaggaggacacaggctc tgtggggctg cagcccagga tgggactaag 7980 tgtgaagaca tctcagcaggtgaggccagg tcccatgaac agagaagcag ctcccacctc 8040 ccctgatgca cggacacacagagtgtgtgg tgctgtgccc ccagagtcgg gctctcctgt 8100 tctggtcccc agggagtgagaagtgaggtt gacttgtccc tgctcctctc tgctacccca 8160 acattcacct tctcctcatgcccctctctc tcaaatatga tttggatcta tgtccccgcc 8220 caaatctcat gtcaaattgtaaaccccaat gttggaggtg gggccttgtg agaagtgatt 8280 ggataatgcg ggtggattttctgctttgat gctgtttctg tgatagagat ctcacatgat 8340 ctggttgttt aaaagtgtgtagcacctctc ccctctctct ctctctctct tactcatgct 8400 ctgccatgta agacgttcctgtttcccctt caccgtccag aatgattgta agttttctga 8460 ggcctcccca ggagcagaagccactatgct tcctgtacaa ctgcagaatg atgagcgaat 8520 taaacctctt ttctttataaattacccagt ctcaggtatt tctttatagc aatgcgagga 8580 cagactaata caatcttctactcccagatc cccgcacacg cttagcccca gacatcactg 8640 cccctgggag catgcacagcgcagcctcct gccgacaaaa gcaaagtcac aaaaggtgac 8700 aaaaatctgc atttggggacatctgattgt gaaagaggga ggacagtaca cttgtagcca 8760 cagagactgg ggctcaccgagctgaaacct ggtagcactt tggcataaca tgtgcatgac 8820 ccgtgttcaa tgtctagagatcagtgttga gtaaaacagc ctggtctggg gccgctgctg 8880 tccccacttc cctcctgtccaccagagggc ggcagagttc ctcccaccct ggagcctccc 8940 caggggctgc tgacctccctcagccgggcc cacagcccag cagggtccac cctcacccgg 9000 gtcacctcgg cccacgtcctcctcgccctc cgagctcctc acacggactc tgtcagctcc 9060 tccctgcagc ctatcggccgcccacctgag gcttgtcggc cgcccacttg aggcctgtcg 9120 gctgccctct gcaggcagctcctgtcccct acaccccctc cttccccggg ctcagctgaa 9180 agggcgtctc ccagggcagctccctgtgat ctccaggaca gctcagtctc tcacaggctc 9240 cgacgccccc tatgctgtcacctcacagcc ctgtcattac cattaactcc tcagtcccat 9300 gaagttcact gagcgcctgtctcccggtta caggaaaact ctgtgacagg gaccacgtct 9360 gtcctgctct ctgtggaatcccagggccca gcccagtgcc tgacacggaa cagatgctcc 9420 ataaatactg gttaaatgtgtgggagatct ctaaaaagaa gcatatcacc tccgtgtggc 9480 ccccagcagt cagagtctgttccatgtgga cacaggggca ctggcaccag catgggagga 9540 ggccagcaag tgcccgcggctgccccagga atgaggcctc aacccccaga gcttcagaag 9600 ggaggacaga ggcctgcagggaatagatcc tccggcctga ccctgcagcc taatccagag 9660 ttcagggtca gctcacaccacgtcgaccct ggtcagcatc cctagggcag ttccagacaa 9720 ggccggaggt ctcctcttgccctccagggg gtgacattgc acacagacat cactcaggaa 9780 acggattccc ctggacaggaacctggcttt gctaaggaag tggaggtgga gcctggtttc 9840 catcccttgc tccaacagacccttctgatc tctcccacat acctgctctg ttcctttctg 9900 ggtcctatga ggaccctgttctgccagggg tccctgtgca actccagact ccctcctggt 9960 accaccatgg ggaaggtggggtgatcacag gacagtcagc ctcgcagaga cagagaccac 10020 ccaggactgt cagggagaacatggacaggc cctgagccgc agctcagcca acagacacgg 10080 agagggaggg tccccctggagccttcccca aggacagcag agcccagagt cacccacctc 10140 cctccaccac agtcctctctttccaggaca cacaagacac ctccccctcc acatgcagga 10200 tctggggact cctgagacctctgggcctgg gtctccatcc ctgggtcagt ggcggggttg 10260 gtggtactgg agacagagggctggtccctc cccagccacc acccagtgag cctttttcta 10320 gcccccagag ccacctctgtcaccttcctg ttgggcatca tcccaccttc ccagagccct 10380 ggagagcatg gggagacccgggaccctgct gggtttctct gtcacaaagg aaaataatcc 10440 ccctggtgtg acagacccaaggacagaaca cagcagaggt cagcactggg gaagacaggt 10500 tgtcctccca ggggatgggggtccatccac cttgccgaaa agatttgtct gaggaactga 10560 aaatagaagg gaaaaaagaggagggacaaa agaggcagaa atgagagggg aggggacaga 10620 ggacacctga ataaagaccacacccatgac ccacgtgatg ctgagaagta ctcctgccct 10680 aggaagagac tcagggcagagggaggaagg acagcagacc agacagtcac agcagccttg 10740 acaaaacgtt cctggaactcaagctcttct ccacagagga ggacagagca gacagcagag 10800 accatggagt ctccctcggcccctccccac agatggtgca tcccctggca gaggctcctg 10860 ctcacaggtg aagggaggacaacctgggag agggtgggag gagggagctg gggtctcctg 10920 ggtaggacag ggctgtgagacggacagagg gctcctgttg gagcctgaat agggaagagg 10980 acatcagaga gggacaggagtcacaccaga aaaatcaaat tgaactggaa ttggaaaggg 11040 gcaggaaaac ctcaagagttctattttcct agttaattgt cactggccac tacgttttta 11100 aaaatcataa taactgcatcagatgacact ttaaataaaa acataaccag ggcatgaaac 11160 actgtcctca tccgcctaccgcggacattg gaaaataagc cccaggctgt ggagggccct 11220 gggaaccctc atgaactcatccacaggaat ctgcagcctg tcccaggcac tggggtgcaa 11280 ccaagatc 11288 5 3774DNA Homo sapiens 5 aagcttttta gtgctttaga cagtgagctg gtctgtctaacccaagtgac ctgggctcca 60 tactcagccc cagaagtgaa gggtgaagct gggtggagccaaaccaggca agcctaccct 120 cagggctccc agtggcctga gaaccattgg acccaggacccattacttct agggtaagga 180 aggtacaaac accagatcca accatggtct ggggggacagctgtcaaatg cctaaaaata 240 tacctgggag aggagcaggc aaactatcac tgccccaggttctctgaaca gaaacagagg 300 ggcaacccaa agtccaaatc caggtgagca ggtgcaccaaatgcccagag atatgacgag 360 gcaagaagtg aaggaaccac ccctgcatca aatgttttgcatgggaagga gaagggggtt 420 gctcatgttc ccaatccagg agaatgcatt tgggatctgccttcttctca ctccttggtt 480 agcaagacta agcaaccagg actctggatt tggggaaagacgtttatttg tggaggccag 540 tgatgacaat cccacgaggg cctaggtgaa gagggcaggaaggctcgaga cactggggac 600 tgagtgaaaa ccacacccat gatctgcacc acccatggatgctccttcat tgctcacctt 660 tctgttgata tcagatggcc ccattttctg taccttcacagaaggacaca ggctagggtc 720 tgtgcatggc cttcatcccc ggggccatgt gaggacagcaggtgggaaag atcatgggtc 780 ctcctgggtc ctgcagggcc agaacattca tcacccatactgacctccta gatgggaatg 840 gcttccctgg ggctgggcca acggggcctg ggcaggggagaaaggacgtc aggggacagg 900 gaggaagggt catcgagacc cagcctggaa ggttcttgtctctgaccatc caggatttac 960 ttccctgcat ctacctttgg tcattttccc tcagcaatgaccagctctgc ttcctgatct 1020 cagcctccca ccctggacac agcaccccag tccctggcccggctgcatcc acccaatacc 1080 ctgataaccc aggacccatt acttctaggg taaggagggtccaggagaca gaagctgagg 1140 aaaggtctga agaagtcaca tctgtcctgg ccagaggggaaaaaccatca gatgctgaac 1200 caggagaatg ttgacccagg aaagggaccg aggacccaagaaaggagtca gaccaccagg 1260 gtttgcctga gaggaaggat caaggccccg agggaaagcagggctggctg catgtgcagg 1320 acactggtgg ggcatatgtg tcttagattc tccctgaattcagtgtccct gccatggcca 1380 gactctctac tcaggcctgg acatgctgaa ataggacaatggccttgtcc tctctcccca 1440 ccatttggca agagacataa aggacattcc aggacatgccttcctgggag gtccaggttc 1500 tctgtctcac acctcaggga ctgtagttac tgcatcagccatggtaggtg ctgatctcac 1560 ccagcctgtc caggcccttc cactctccac tttgtgaccatgtccaggac cacccctcag 1620 atcctgagcc tgcaaatacc cccttgctgg gtgggtggattcagtaaaca gtgagctcct 1680 atccagcccc cagagccacc tctgtcacct tcctgctgggcatcatccca ccttcacaag 1740 cactaaagag catggggaga cctggctagc tgggtttctgcatcacaaag aaaataatcc 1800 cccaggttcg gattcccagg gctctgtatg tggagctgacagacctgagg ccaggagata 1860 gcagaggtca gccctaggga gggtgggtca tccacccaggggacaggggt gcaccagcct 1920 tgctactgaa agggcctccc caggacagcg ccatcagccctgcctgagag ctttgctaaa 1980 cagcagtcag aggaggccat ggcagtggct gagctcctgctccaggcccc aacagaccag 2040 accaacagca caatgcagtc cttccccaac gtcacaggtcaccaaaggga aactgaggtg 2100 ctacctaacc ttagagccat caggggagat aacagcccaatttcccaaac aggccagttt 2160 caatcccatg acaatgacct ctctgctctc attcttcccaaaataggacg ctgattctcc 2220 cccaccatgg atttctccct tgtcccggga gccttttctgccccctatga tctgggcact 2280 cctgacacac acctcctctc tggtgacata tcagggtccctcactgtcaa gcagtccaga 2340 aaggacagaa ccttggacag cgcccatctc agcttcacccttcctccttc acagggttca 2400 gggcaaagaa taaatggcag aggccagtga gcccagagatggtgacaggc agtgacccag 2460 gggcagatgc ctggagcagg agctggcggg gccacagggagaaggtgatg caggaaggga 2520 aacccagaaa tgggcaggaa aggaggacac aggctctgtggggctgcagc ccagggttgg 2580 actatgagtg tgaagccatc tcagcaagta aggccaggtcccatgaacaa gagtgggagc 2640 acgtggcttc ctgctctgta tatggggtgg gggattccatgccccataga accagatggc 2700 cggggttcag atggagaagg agcaggacag gggatccccaggataggagg accccagtgt 2760 ccccacccag gcaggtgact gatgaatggg catgcagggtcctcctgggc tgggctctcc 2820 ctttgtccct caggattcct tgaaggaaca tccggaagccgaccacatct acctggtggg 2880 ttctggggag tccatgtaaa gccaggagct tgtgttgctaggaggggtca tggcatgtgc 2940 tgggggcacc aaagagagaa acctgagggc aggcaggacctggtctgagg aggcatggga 3000 gcccagatgg ggagatggat gtcaggaaag gctgccccatcagggagggt gatagcaatg 3060 gggggtctgt gggagtgggc acgtgggatt ccctgggctctgccaagttc cctcccatag 3120 tcacaacctg gggacactgc ccatgaaggg gcgcctttgcccagccagat gctgctggtt 3180 ctgcccatcc actaccctct ctgctccagc cactctgggtctttctccag atgccctgga 3240 cagccctggc ctgggcctgt cccctgagag gtgttgggagaagctgagtc tctggggaca 3300 ctctcatcag agtctgaaag gcacatcagg aaacatccctggtctccagg actaggcaat 3360 gaggaaaggg ccccagctcc tccctttgcc actgagagggtcgaccctgg gtggccacag 3420 tgacttctgc gtctgtccca tgcaccctga aaccacaacaaaaccccagc cccagaccct 3480 gcaggtacaa tacatgtggg gacagtctgt acccaggggaagccagttct ctcttcctag 3540 gagaccgggc ctcagggctg tgcccggggc aggcgggggcagcacgtgcc tgtccttgag 3600 aactcgggac cttaagggtc tctgctctgt gaggcacagcaaggatcctt ctgtccagag 3660 atgaaagcag ctcctgcccc tcctctgacc tcttcctccttcccaaatct caaccaacaa 3720 ataggtgttt caaatctcat catcaaatct tcatccatccacatgagaaa gctt 3774 6 1634 DNA Escherichia coli 6 ctgcaggcca ctggttaccgggaattgttc cggtcaacgc ggtattaggt ggcgcgctga 60 gctatctgat ccttaacccgattttgaatc gtaaaacgac agcagcaatg acgcatgtgg 120 aggctaacag tgtcgaataacgctttacaa acaattatta acgcccggtt accaggcgaa 180 gaggggctgt ggcagattcatctgcaggac ggaaaaatca gcgccattga tgcgcaatcc 240 ggcgtgatgc ccataactgaaaacagcctg gatgccgaac aaggtttagt tataccgccg 300 tttgtggagc cacatattcacctggacacc acgcaaaccg ccggacaacc gaactggaat 360 cagtccggca cgctgtttgaaggcattgaa cgctgggccg agcgcaaagc gttattaacc 420 catgacgatg tgaaacaacgcgcatggcaa acgctgaaat ggcagattgc caacggcatt 480 cagcatgtgc gtacccatgtcgatgtttcg gatgcaacgc taactgcgct gaaagcaatg 540 ctggaagtga agcaggaagtcgcgccgtgg attgatctgc aaatcgtcgc cttccctcag 600 gaagggattt tgtcgtatcccaacggtgaa gcgttgctgg aagaggcgtt acgcttaggg 660 gcagatgtag tgggggcgattccgcatttt gaatttaccc gtgaatacgg cgtggagtcg 720 ctgcataaaa ccttcgccctggcgcaaaaa tacgaccgtc tcatcgacgt tcactgtgat 780 gagatcgatg acgagcagtcgcgctttgtc gaaaccgttg ctgccctggc gcaccatgaa 840 ggcatgggcg cgcgagtcaccgccagccac accacggcaa tgcactccta taacggggcg 900 tatacctcac gcctgttccgcttgctgaaa atgtccggta ttaactttgt cgccaacccg 960 ctggtcaata ttcatctgcaaggacgtttc gatacgtatc caaaacgtcg cggcatcacg 1020 cgcgttaaag agatgctggagtccggcatt aacgtctgct ttggtcacga tgatgtcttc 1080 gatccgtggt atccgctgggaacggcgaat atgctgcaag tgctgcatat ggggctgcat 1140 gtttgccagt tgatgggctacgggcagatt aacgatggcc tgaatttaat cacccaccac 1200 agcgcaagga cgttgaatttgcaggattac ggcattgccg ccggaaacag cgccaacctg 1260 attatcctgc cggctgaaaatgggtttgat gcgctgcgcc gtcaggttcc ggtacgttat 1320 tcggtacgtg gcggcaaggtgattgccagc acacaaccgg cacaaaccac cgtatatctg 1380 gagcagccag aagccatcgattacaaacgt tgaacgactg ggttacagcg agcttagttt 1440 atgccggatg cggcgtgaacgccttatccg gcctacgtag agcactgaac tcgtaggcct 1500 gataagcgta gcgcatcaggcaattccagc cgctgatctg tgtcagcggc taccgtgatt 1560 cattcccgcc aacaaccgcgcattcctcca acgccatgtg caaaaatgcc ttcgcagcgg 1620 ctgtctgcca gctg 1634 740 DNA Artificial sequence misc_feature Description of artificialsequence oligonucleotide to hybridize with human CE 7 ccctgtgatctccaggacag ctcagtctcc gtccaatctc 40 8 28 DNA Artificial sequencemisc_feature Description of artificial sequence oligonucleotide tohybridize with human CE 8 gtttcctgag tgatgtctgt gtgcaatg 28 9 35 DNAArtificial sequence misc_feature Description of artificial sequenceoligonucleotide to introduce restriction site 9 cctggaactc aagcttgaattctccacaga ggagg 35 10 34 DNA Artificial sequence misc_featureDescription of artificial sequence oligonucleotide to isolate cy tosinedeaminas 10 gacgcatgtg gaagcttaca atgtcgaata acgc 34 11 341 PRTVaricella zoster 11 Met Ser Thr Asp Lys Thr Asp Val Lys Met Gly Val LeuArg Ile Tyr 1 5 10 15 Leu Asp Gly Ala Tyr Gly Ile Gly Lys Thr Thr AlaAla Glu Glu Phe 20 25 30 Leu His His Phe Ala Ile Thr Pro Asn Arg Ile LeuLeu Ile Gly Glu 35 40 45 Pro Leu Ser Tyr Trp Arg Asn Leu Ala Gly Glu AspAla Ile Cys Gly 50 55 60 Ile Tyr Gly Thr Gln Thr Arg Arg Leu Asn Gly AspVal Ser Pro Glu 65 70 75 80 Asp Ala Gln Arg Leu Thr Ala His Phe Gln SerLeu Phe Cys Ser Pro 85 90 95 His Ala Ile Met His Ala Lys Ile Ser Ala LeuMet Asp Thr Ser Thr 100 105 110 Ser Asp Leu Val Gln Val Asn Lys Glu ProTyr Lys Ile Met Leu Ser 115 120 125 Asp Arg His Pro Ile Ala Ser Thr IleCys Phe Pro Leu Ser Arg Tyr 130 135 140 Leu Val Gly Asp Met Ser Pro AlaAla Leu Pro Gly Leu Leu Phe Thr 145 150 155 160 Leu Pro Ala Glu Pro ProGly Thr Asn Leu Val Val Cys Thr Val Ser 165 170 175 Leu Pro Ser His LeuSer Arg Val Ser Lys Arg Ala Arg Pro Gly Glu 180 185 190 Thr Val Asn LeuPro Phe Val Met Val Leu Arg Asn Val Tyr Ile Met 195 200 205 Leu Ile AsnThr Ile Ile Phe Leu Lys Thr Asn Asn Trp His Ala Gly 210 215 220 Trp AsnThr Leu Ser Phe Cys Asn Asp Val Phe Lys Gln Lys Leu Gln 225 230 235 240Lys Ser Glu Cys Ile Lys Leu Arg Glu Val Pro Gly Ile Glu Asp Thr 245 250255 Leu Phe Ala Val Leu Lys Leu Pro Glu Leu Cys Gly Glu Phe Gly Asn 260265 270 Ile Leu Pro Leu Trp Ala Trp Gly Met Glu Thr Leu Ser Asn Cys Leu275 280 285 Arg Ser Met Ser Pro Phe Val Leu Ser Leu Glu Gln Thr Pro GlnHis 290 295 300 Ala Ala Gln Glu Leu Lys Thr Leu Leu Pro Gln Met Thr ProAla Asn 305 310 315 320 Met Ser Ser Gly Ala Trp Asn Ile Leu Lys Glu LeuVal Asn Ala Val 325 330 335 Gln Asp Asn Thr Ser 340 12 42 PRT Varicellazoster 12 Met Ser Thr Asp Lys Thr Asp Val Lys Met Gly Val Leu Arg IleTyr 1 5 10 15 Leu Asp Gly Ala Tyr Gly Ile Gly Lys Thr Thr Ala Ala GluGlu Phe 20 25 30 Leu His His Phe Ala Ile Thr Pro Asn Arg 35 40 13 42 PRTVaricella zoster 13 Met Ser Thr Asp Lys Thr Asp Val Lys Met Gly Val LeuArg Ile Tyr 1 5 10 15 Leu Asp Gly Ala Tyr Gly Ile Gly Lys Thr Thr AlaAla Glu Glu Phe 20 25 30 Leu His His Phe Ala Ile Thr Pro Asn Arg 35 4014 21 PRT Homo sapiens 14 Met Glu Ser Pro Ser Ala Pro Pro His Arg TrpCys Ile Pro Trp Gln 1 5 10 15 Arg Leu Leu Leu Thr 20 15 427 PRTEscherichia coli 15 Val Ser Asn Asn Ala Leu Gln Thr Ile Ile Asn Ala ArgLeu Pro Gly 1 5 10 15 Glu Glu Gly Leu Trp Gln Ile His Leu Gln Asp GlyLys Ile Ser Ala 20 25 30 Ile Asp Ala Gln Ser Gly Val Met Pro Ile Thr GluAsn Ser Leu Asp 35 40 45 Ala Glu Gln Gly Leu Val Ile Pro Pro Phe Val GluPro His Ile His 50 55 60 Leu Asp Thr Thr Gln Thr Ala Gly Gln Pro Asn TrpAsn Gln Ser Gly 65 70 75 80 Thr Leu Phe Glu Gly Ile Glu Arg Trp Ala GluArg Lys Ala Leu Leu 85 90 95 Thr His Asp Asp Val Lys Gln Arg Ala Trp GlnThr Leu Lys Trp Gln 100 105 110 Ile Ala Asn Gly Ile Gln His Val Arg ThrHis Val Asp Val Ser Asp 115 120 125 Ala Thr Leu Thr Ala Leu Lys Ala MetLeu Glu Val Lys Gln Glu Val 130 135 140 Ala Pro Trp Ile Asp Leu Gln IleVal Ala Phe Pro Gln Glu Gly Ile 145 150 155 160 Leu Ser Tyr Pro Asn GlyGlu Ala Leu Leu Glu Glu Ala Leu Arg Leu 165 170 175 Gly Ala Asp Val ValGly Ala Ile Pro His Phe Glu Phe Thr Arg Glu 180 185 190 Tyr Gly Val GluSer Leu His Lys Thr Phe Ala Leu Ala Gln Lys Tyr 195 200 205 Asp Arg LeuIle Asp Val His Cys Asp Glu Ile Asp Asp Glu Gln Ser 210 215 220 Arg PheVal Glu Thr Val Ala Ala Leu Ala His His Glu Gly Met Gly 225 230 235 240Ala Arg Val Thr Ala Ser His Thr Thr Ala Met His Ser Tyr Asn Gly 245 250255 Ala Tyr Thr Ser Arg Leu Phe Arg Leu Leu Lys Met Ser Gly Ile Asn 260265 270 Phe Val Ala Asn Pro Leu Val Asn Ile His Leu Gln Gly Arg Phe Asp275 280 285 Thr Tyr Pro Lys Arg Arg Gly Ile Thr Arg Val Lys Glu Met LeuGlu 290 295 300 Ser Gly Ile Asn Val Cys Phe Gly His Asp Asp Val Phe AspPro Trp 305 310 315 320 Tyr Pro Leu Gly Thr Ala Asn Met Leu Gln Val LeuHis Met Gly Leu 325 330 335 His Val Cys Gln Leu Met Gly Tyr Gly Gln IleAsn Asp Gly Leu Asn 340 345 350 Leu Ile Thr His His Ser Ala Arg Thr LeuAsn Leu Gln Asp Tyr Gly 355 360 365 Ile Ala Ala Gly Asn Ser Ala Asn LeuIle Ile Leu Pro Ala Glu Asn 370 375 380 Gly Phe Asp Ala Leu Arg Arg GlnVal Pro Val Arg Tyr Ser Val Arg 385 390 395 400 Gly Gly Lys Val Ile AlaSer Thr Gln Pro Ala Gln Thr Thr Val Tyr 405 410 415 Leu Glu Gln Pro GluAla Ile Asp Tyr Lys Arg 420 425 16 6 PRT Consensus sequence misc_featureConsensus sequence A1 from transcriptional dictionary of Locker a ndBuzard (1990). 16 Thr Ala Thr Ala Trp Trp 1 5 17 15 PRT Consensussequence misc_feature Consensus sequence A2calt from transcriptionaldictionary of Lock er and Buzard (1990). 17 Thr Thr Gly Gly Cys Asn AsnAsn Asn Asn Asn Gly Cys Cys Ala 1 5 10 15 18 14 PRT Consensus sequencemisc_feature Consensus sequence A4alt from transcriptional dictionary ofLock er and Buzard (1990). 18 Arg Arg Arg Asn Cys Cys His Cys Ala CysCys Cys Thr Gly 1 5 10 19 8 PRT Consensus sequence misc_featureConsensus sequence B2 from transcriptional dictionary of Locker a ndBuzard (1990). 19 Gly Thr Gly Gly Trp Trp Trp Gly 1 5 20 8 PRT Consensussequence misc_feature Consensus sequence B4 from transcriptionaldictionary of Locker a nd Buzard (1990). 20 Gly Ser Ser Trp Gly Ser CysCys 1 5 21 10 PRT Consensus sequence misc_feature Consensus sequence B12from transcriptional dictionary of Locker and Buzard (1990). 21 Cys CysTrp Trp Trp Trp Trp Trp Gly Gly 1 5 10 22 6 PRT Consensus sequencemisc_feature Consensus sequence B15 from transcriptional dictionary ofLocker and Buzard (1990). 22 Gly Ala Ala Ala Gly Tyr 1 5 23 6 PRTConsensus sequence misc_feature Consensus sequence B17 fromtranscriptional dictionary of Locker and Buzard (1990). 23 Thr Cys MetTyr Thr Thr 1 5 24 9 PRT Consensus sequence misc_feature Consensussequence B18 from transcriptional dictionary of Locker and Buzard(1990). 24 Ala Asn Cys Cys Thr Cys Thr Cys Tyr 1 5 25 8 PRT Consensussequence misc_feature Consensus sequence C5 from transcriptionaldictionary of Locker a nd Buzard (1990). 25 Gly Thr Gly Ser Gly Gly ThrGly 1 5 26 8 PRT Consensus sequence misc_feature Consensus sequence D9from transcriptional dictionary of Locker a nd Buzard (1990). 26 Arg ThrGly Ala Cys Gly Thr Arg 1 5 27 12 PRT Consensus sequence misc_featureConsensus sequence E5 from transcriptional dictionary of Locker a ndBuzard (1990). 27 Ala Cys Cys Asn Asn Asn Asn Asn Asn Gly Gly Thr 1 5 1028 6 PRT Consensus Sequence misc_feature Consensus sequence F2 fromtranscriptional dictionary of Locker a nd Buzard (1990). 28 Thr Gly ArgMet Cys Cys 1 5 29 8 PRT Consensus sequence misc_feature Consensussequence F6 from transcriptional dictionary of Locker a nd Buzard(1990). 29 Thr Cys Asn Thr Ala Cys Thr Cys 1 5 30 8 PRT ConsensusSequence misc_feature Consensus sequence F7 from transcriptionaldictionary of Locker a nd Buzard (1990). 30 Thr Gly Thr Thr Thr Gly CysThr 1 5 31 5 PRT Consensus sequence misc_feature Consensus sequence F9from transcriptional dictionary of Locker a nd Buzard (1990). 31 Thr CysAla Cys Thr 1 5 32 9 PRT Consensus Sequence misc_feature Consensussequence F10 from transcriptional dictionary of Locker and Buzard(1990). 32 Trp Thr Ser Thr Gly Gly Gly Ala Trp 1 5 33 8 PRT Consensussequence misc_feature Consensus sequence G2 from transcriptionaldictionary of Locker a nd Buzard (1990). 33 Ala Ala Asn Cys Cys Ala AlaAla 1 5 34 6 PRT Consensus sequence misc_feature Consensus sequence G7from transcriptional dictionary of Locker a nd Buzard (1990). 34 Gly AlaThr Ala Ala Gly 1 5 35 17 PRT Consensus sequence misc_feature Consensussequence H1 from transcriptional dictionary of Locker a nd Buzard(1990). 35 Arg Asn Tyr Asn Asn Cys Asn Asn Gly Tyr Asn Gly Lys Thr AsnTyr 1 5 10 15 Asn 36 16 PRT Consensus sequence misc_feature Consensussequence 90% H1 from transcriptional dictionary of Lock er and Buzard(1990). 36 Arg Tyr Asn Asn Cys Asn Asn Gly Tyr Asn Gly Lys Thr Asn TyrAsn 1 5 10 15

We claim:
 1. A molecular chimaera comprising a transcriptionalregulatory sequence capable of being selectively activated in amammalian target tissue, a DNA sequence operatively linked to thetranscriptional regulatory sequence and encoding a heterologous enzyme,the enzyme capable of catalysing the production of an agent toxic to thetarget tissue.
 2. A chimaera as claimed in claim 1 further comprising apromoter wherein said promoter is selected from the promoters ofalbumin, alpha-fetoprotein, carcinoembryonic antigen, tyrosinehydroxylase, choline acetyl transferase, neuron-specific enolase, glialfibro acidic protein, insulin, gama-glutamyltranspeptidase, dopadecarboxylase, HER-2/neu and N-myc oncogenes.
 3. A chimaera as claimedin claim 1 further comprising an enhancer wherein the enhancer isselected from the enhancers of albumin, alpha-fetoprotein,carcino-embryonic antigen, tyrosine hydroxylase, choline acetyltransferase, glial fibro acidic protein, insulin,gama-glutamyltranspeptidase, dopa decarboxylase, HER-2/neu andN-myconcogenes.
 4. A molecular chimaera as claimed in claim 1 wherein the DNAsequence encoding a heterologous enzyme is selected from; varicellazoster virus thymidine kinase (VZVTK); carboxypeptidase G2; alkalinephosphatase; penicillin-V amidase; and cytosine deaminase.
 5. A methodof treating cancerous cells comprising administering to said cells, aDNA sequence capable of being selectively activated in said cells toexpress an enzyme, and administering an agent which is converted in saidcells by said enzyme to an agent which is cytotoxic or cytostatic tosaid cancer cells.
 6. A method of treating cancerous host cellscomprising administering to a host an infective virion encapsidating aretroviral shuttle vector comprising a molecular chimaera, said chimaeracomprising a transcriptional regulatory sequence which is selectivelyactivated in cancerous cells and operatively linked to a gene encoding aheterologous enzyme; in an amount sufficient to transform said canceroushost cells so as to express said enzyme, and administering to the hostan amount of a compound which is selectively metabolised in said hostcells by said enzyme to a cytotoxic or cytostatic metabolite.
 7. Amolecular chimaera comprising a transcriptional regulatory sequenceselected from the group consisting of albumin, alpha-fetoprotein, andcarcinoembryonic antigen; said transcriptional regulatory sequencecapable of being selectively activated in cancerous cells; a DNAsequence operatively linked to the transcriptional regulatory sequenceand encoding a heterologous enzyme selected from the group consisting ofVZVTK, cytosine deaminase, carboxypeptidase G2, alkaline phosphatase andpenicillin-V amidase, which are capable of catalyzing the production ofan agent cytotoxic or cytostatic to the cancerous cells.
 8. A molecularchimaera comprising a transcriptional regulatory sequence selected fromthe group consisting of albumin, alpha-fetoprotein or carcinoembryonicantigen; said transcriptional regulatory sequence capable of beingselectively activated in cancerous cells; said cancerous cells selectedfrom the group consisting of hepatoma, colorectal carcinoma, metastaticcolorectal carcinoma, and hepatic colorectal carcinoma; a DNA sequenceoperatively linked to the transcriptional regulatory sequence andencoding cytosine deaminase which is capable of catalyzing theproduction of an agent cytotoxic or cytostatic to the cancerous cells.9. The chimaera as claimed in claims 7 or 8 wherein the transcriptionalregulatory sequence comprises a promoter selected from the promoters ofalbumin, alpha-fetoprotein, and carcinoembryonic antigen.
 10. Thechimaera as claimed in claim 9 wherein the transcriptional regulatorysequence additionally comprises an enhancer selected from the enhancersof albumin, aipha-fetoprotein, and carcinoembryonic antigen.
 11. Thechimaera as claimed in claim 8 additionally comprising a polyadenylationsignal downstream of the DNA sequence encoding cytosine deaminase. 12.The molecular chimaera as claimed in claim 8 wherein the cytosinedeaminase is non-mammalian cytosine deaminase.
 13. The molecularchimaera as claimed in claim 12 wherein the non-mammalian cytosinedeaminase is selected from the group consisting of bacterial cytosinedeaminase, fungal cytosine deaminase and yeast cytosine deaminase. 14.The molecular chimaera as claimed in claim 13 wherein the bacterialcytosine deaminase is E. coli cytosine deaminase.
 15. The molecularchimaera of claim 8 wherein the cytosine deaminase is capable ofcatalyzing the production of 5-fluorouracil from 5-fluorocytosine. 16.The molecular chimaera of claim 15 wherein the 5-fluorouracil diffusesout of the cancerous cell.
 17. A molecular chimaera comprising a DNAsequence encoding the gene for E. coli cytosine deaminase operativelylinked to the transcriptional regulatory sequence for carcinoembryonicantigen.
 18. A gene delivery system comprising a molecular chimaera,said chimaera comprising a transcriptional regulatory sequence forcarcinoembryonic antigen which is selectively activated in cancerouscells and operatively linked to the coding sequence for the geneencoding cytosine deaminase.
 19. The gene delivery system of claim 18which is selected from a viral, non-viral or a receptor-mediateddelivery system.
 20. The gene delivery system of claim 19 wherein theviral delivery system is retroviral or adenoviral shuttle vector. 21.The gene delivery system of claim 19 wherein the non-viral deliverysystem is a liposomal delivery system.
 22. An infective virioncomprising a retroviral shuttle vector, said vector comprising amolecular chimaera, said chimaera comprising a transcriptionalregulatory sequence for carcinoembryonic antigen which is selectivelyactivated in cancerous cells and operatively linked to a gene encodingcytosine deaminase.
 23. A packaging cell line comprising a retroviral oradenoviral shuttle vector said shuttle vector further comprising amolecular chimaera.
 24. The packaging cell line of claim 23 wherein themolecular chimaera comprises a transcriptional regulatory sequence forcarcinoembryonic antigen which is selectively activated in cancerouscells and operatively linked to a gene encoding cytosine deaminase. 25.A method of treating a host in need of anticancer treatment comprisingadministering to said host, a molecular chimaera capable of beingselectively activated in cancerous cells of said host to express theenzyme cytosine deaminase, and subsequently administering to said host5-fluorocytosine which is converted in said cells by said enzyme to5-fluorouracil which is cytotoxic or cytostatic to said cells.
 26. Amethod of treating a host in need of anticancer treatment comprisingadministering to said host an infective virion encapsidating aretroviral or adenoviral shuttle vector comprising a molecular chimaera,said chimaera comprising a transcriptional regulatory sequence which isselectively activated in cancerous cells of said host and operativelylinked to a gene encoding the heterologous enzyme cytosine deaminase; inan amount sufficient to transform said cells so as to express saidenzyme, and subsequently administering to the host an amount of5-fluorocytosine which is selectively metabolised in said cells by saidenzyme to 5-fluorouracil.
 27. A method of treating a host in need ofanticancer treatment comprising administering to said host a packagingcell line, said packaging cell line further comprising a retroviral oradenoviral shuttle vector capable of producing an infective virion, saidshuttle vector comprising a molecular chimaera, said chimaera comprisinga transcriptional regulatory sequence which is selectively activated incancerous cells of said host and operatively linked to a gene encodingthe heterologous enzyme cytosine deaminase; in an amount sufficient totransform said cells so as to express said enzyme, and subsequentlyadministering to the host an amount of 5-fluorocytosine which isselectively metabolised in said cells by said enzyme to 5-fluorouracil.28. The method of claim 25, 26 or 27 wherein the host is a mammal. 29.The method of claim 25, 26 or 27 wherein the host is a human.
 30. Themethod as claimed in claim 25, 26 or 27 wherein the 5-fluorocytosine ismetabolised by the enzyme to an intermediate, which in turn is convertedto 5-fluorouracil.
 31. The method as claimed in claim 25, 26 or 27 whichfurther comprises administering to said host a 5-fluorouracilpotentiating agent or an agent which is metabolised to a 5-fluorouracilpotentiating agent in combination with said 5-fluorocytosine.
 32. Themethod of claim 31 wherein the 5-fluorouracil potentiating agent isleucovorin, levemisol, 5-ethynyluracil or 5-bromovinyluracil.
 33. Amethod of constructing a molecular chimaera comprising linking a DNAsequence encoding cytosine deaminase to a tissue-specific promoterselected from the group consisting of ALB, AFP or CEA.
 34. A method ofkilling or arresting the growth of cells comprising delivering amolecular chimaera into said cells, said chimaera expressing aheterologous enzyme in said cells and exposing said cells to an agentwhich is converted by said enzyme to an agent which is cytotoxic orcytostatic to said cells.
 35. The method of claim 34 wherein saidheterologous enzyme is cytosine deaminase, said agent which is convertedby said enzyme is 5-fluorouracil and said cytotoxic or cytostatic agentis 5-fluorouracil.
 36. A pharmaceutical composition comprising aninfective virion as claimed in claim 16 in admixture with apharmaceutically acceptable carrier.
 37. A pharmaceutical compositioncomprising a packaging cell line as claimed in claim 18 in admixturewith a pharmaceutically acceptable carrier.
 38. A plasmid selected fromthe group consisting of pCR92, pCR105, pCR113, pCR136, pCR137, pCR145,pCR148, pCR158, pCR162, pCR163, pEA001, pEA002, pEA003, 39-5-2 and39-5-5.
 39. The clone BA101.
 40. The isolated DNA of SEQ ID NO:4.